Antibiotic acanthomycin and process for preparing

BRIEF SUMMARY OF THE INVENTION
The antibiotic of the invention, acanthomycin, is obtained by culturing the novel microorgaism Streptomyces espinosus subsp. acanthus, NRRL 11081, in an aqueous nutrient medium under aerobic conditions. Acanthomycin and its base addition salts have the property of adversely affecting the growth of Gram-positive bacteria, for example, Staphylococcus aerueus and Streptococcus hemolyticus. Accordingly, acanthomycin and its base addition salts can be used alone or in combination with other antibiotic agents to prevent the growth of or reduce the number of bacteria, as disclosed above, in various environments.
DETAILED DESCRIPTION OF THE INVENTION
Chemical and Physical Properties of Acanthomycin
Empirical Formula: (C.sub.90 H.sub.139 N.sub.19 O.sub.42).sub.n ; Equivalent weight, 2,159.
Elemental Analysis:
Calcd.: C, 50.06; H, 6.48; N, 12.33; O, 31.12. Found: C, 49.81; H, 6.47; N, 12.36; O (by difference), 31.36.
Optical Rotation: [.alpha.].sub.D.sup.25, -28.5.degree. (c, 1, dimethylsulfoxide).
Solubilities: Acanthomycin free acid is soluble in dimethylformamide and dimethylsulfoxide. It is slightly soluble (ca. 1-2 mg/ml) in methanol and ethanol, and relatively insoluble in water, ether, halogenated hydrocarbon and saturated hydrocarbon solvents. Salts (ammonium and sodium) are soluble in water and lower alcohols.
Titration Data
Equivalent weight: 2,159
Titrant: KOH
Solvent: Dimethylformamide-60% aqueous ethanol (1:1).
Thin Layer Chromatography: On silica gel (Merck & Co.) using chloroform-ethanol-water (2:3:1 v/v), R.sub.f =0.52.
Infrared Absorption Spectra: Acanthomycin has a characteristic infrared absorption spectrum in a mineral oil mull as shown in FIG. 1 of the drawings. Peaks are observed at the following wave lengths expressed in reciprocal centimeters:
______________________________________Band Frequency(Wave Numbers) Intensity______________________________________3280 S3070 M2960 S2930 S3860 S2650 W1723 M1656 S, sh (sh = shoulder)1643 S1540 S1463 S1415 M1378 M1341 M1278 M1268 M1240 M1223 M1145 W, sh1187 M1098 W1065 W1033 W1002 W 960 W 943 W 920 W 847 W 723 W 699 M______________________________________ Key: S = Strong, M = Medium, and W = Weak
Acanthomycin also has a characteristic infrared absorption spectrum when pressed in a KBr disc. Peaks are observed at the following wave lengths expressed in reciprocal centimeters:
______________________________________Band Frequency(Wave Numbers) Intensity______________________________________3390 S, sh3290 S3060 M2960 M2930 M2880 M2650 W1724 M, sh1652 S1533 S1455 M1412 M1385 M1340 M1273 M1223 M1183 M1140 M, sh1095 W1067 W1030 W1000 W 960 W 920 W 875 W 737 W 698 M______________________________________
Ultraviolet Absorption Spectrum: Acanthomycin has a characteristic ultraviolet absorption spectrum in methanol as shown in FIG. 2 of the drawings. .lambda. max at 262 (a=16.81) in methanol.
BIOLOGICAL PROPERTIES OF ACANTHOMYCIN
Acanthomycin was tested for antibacterial activity in a standard tube assay using nutrient broth as the medium. The minimum inhibitory concentration in mcg/ml against S. aureus was 50, and against S. lutea it was 25. Additional testing on a standard disc (12.7 mm) plate assay gave the following results:
______________________________________ Zone (mm)Acanthomycin Strepto- Staphylo- Sar-mg/ml coccus coccus Bacillus Bacillus cinaConcentration pyogenes aureus cereus subtilis lutea______________________________________10 41.5 29.5 26 34 355 40 28.5 25 33 342.5 40 28 23 31.5 321.25 38.5 26.5 21 30 300.62 36.5 24.5 20 28 270.31 34 22.5 17 26 25______________________________________
Acanthomycin was also tested in vivo in standard laboratory mice. S. pyogenese infected mice were protected by acanthomycin administered subcutaneously with a CD.sub.50 of 0.23 (0.17-0.31) mg/kg. Against S. aureus infected mice the CD.sub.50 was 1.0 mg/kg.
THE MICROORGANISM
The microorganism used for the production of acanthomycin is Streptomyces espinosus subsp. acanthus, NRRL 11081. A subculture of this microorganism is freely available from the permanent collection of the Northern Regional Research Laboratory, U.S. Department of Agriculture, Peoria, Ill., U.S.A. by request made thereto. It should be understood that the availability of the culture does not constitute a license to practice the subject invention in derogation of patent rights granted with the subject instrument by governmental action.
The microorganism of this invention was studied and characterized by Alma Dietz of the Upjohn Research Laboratories.
An actinomycete culture, isolated from a soil sample from southeast Texas was characterized as Streptomyces espinosus sp. n. in U.S. Pat. No. 3,697,380. Four additional soil isolates from southeast Texas soils had the same color pattern on Ektachrome as the type culture. In expanded studies the isolates were found to have the same sporophore and spore types as well as general cultural characteristics of the type culture. Outstanding characteristics of these cultures are their gray-green aerial growth, short sporophores bearing round spiny spores, and thermoduric growth. Differences in cultural characteristics (see Tables) and antagonistic properties are not sufficient to warrant variety designation of these new isolates. Three of these new isolates, which are disclosed in U.S. Pat. No. 3,833,475, have the accession numbers NRRL 5729, NRRL 5730 and NRRL 5731. The remaining isolate is described herein as Stroptomyces espinosus subsp. acanthus, NRRL 11081.
Methods for culture characterization were those cited by Dietz [Dietz, A. 1967. Streptomyces steffisburgensis sp. n. J. Bacteriol. 94: 2,022-2,026.] and in part those cited by Shirling and Gottlieb [Shirling, E. B., and D. Gottlieb. 1966. Methods for characterization of Streptomyces species. Int. J. Syst. Bacteriol. 16: 313-340.]. The cultures were also grown on Hickey-Tresner agar [Hickey, R. J. and H. D. Tresner. 1952. A cobalt-containing medium for sporulation of Streptomyces species. J. Bacteriol. 64: 891-892.] modified [Pepticase (enzymatic digest of casein) substituted for N-Z Amine A].
Color characteristics: Aerial growth gray-green. Melanin-negative. Appearance on Ektachrome [Dietz, A. 1954. Ektachrome transparencies as aids in actinomycete classification. Ann. N.Y. Acad. Sci. 60: 152-154.] is given in Table 1. Reference color characteristics are given in Tables 2 and 3. The cultures may be placed in the Green (GN) color series of Tresner and Backus [Tresner, H. D., and E. J. Backus. 1963. System of color wheels for streptomycete taxonomy. Applied Microbiol. 11: 335-338.].
Microscopic characteristics: Sporophores short, straight to flexuous, to open spiral to spiral (RF, RA, S) in the sense of Pridham et al. [Pridham, T. G., C. W. Hesseltine, and R. G. Benedict. 1958. A guide for the classification of streptomycetes according to selected groups. Placement of strains in morphological sections. Applied Microbiol. 6: 52-79.]. Spores mostly spherical with many showing a distinct linkage. Spore surface thorny to spiny in the sense of Dietz and Mathews [Dietz, A., and J. Mathews. 1971. Classification of Streptomyces spore surfaces into five groups. Applied Microbiol. 21: 527-533.]. Some spines show a transition to hairy. Spines are profuse and show markings when observed on spores treated by the carbon replica method of Dietz and Mathews [Dietz, A., and J. Mathews. 1962. Taxonomy by carbon replication. I. An examination of Streptomyces hygroscopicus. Applied Microbiol. 10: 258-263.].
Cultural and biochemical characteristics: Cultural and biochemical characteristics are cited in Table 4.
Carbon utilization: Growth of the cultures on carbon compounds was determined in the synthetic medium of Pridham and Gottlieb [Pridham, T. G., and D. Gottlieb. 1948. The utilization of carbon compounds by some Actinomycetales as an aid for species determination. J. Bacteriol. 56: 107-114.], Table 5 and in the synthetic medium of Shirling and Gottlieb, op. cit., 313-340, Table 6.
Temperature: Growth was fair at 18.degree. C. and 55.degree. C.; good at 24.degree. C., and heavy at 28.degree.-37.degree. C. At 45.degree. C. growth was fair (vegative) in 24 hours and heavy (good sporulation) in 72 hours. The agar media used were Bennett's, Czapek's sucrose, maltose-tryptone, and Hickey-Tresner (modified).
Antibiotic-producing properties: S. espinosus NRRL 5729, NRRL 5730, and NRRL 5731 produce the antibiotic lincomycin. S. espinosus subsp. acanthus, NRRL 11081, produces acanthomycin.
TABLE 1__________________________________________________________________________Appearance of Streptomyces espinosus Cultures on Ektachrome* De- ter- mi-Agar na- NRRL NRRL NRRL NRRL NRRLMedium tion 3890 5729 5730 5731 11081__________________________________________________________________________Bennett's S Gray-green Gray-green White to gray- Gray-green Gray-green green R Pale yellow-tan Pale yellow-tan Pale yellow Pale yellow-tan Pale yellowCzapek's S Gray-green Gray-green Gray-green Gray-green Gray-greensucrose R Pale gray Pale gray Pale gray Pale grayMaltose- S Gray-green Gray-green Gray-green Gray-green Gray-greentryptone R Yellow-tan to Yellow-tan to Yellow Yellow-tan Pale yellow- olive olive tanPeptone-iron S White White White White White R Yellow Yellow Yellow Yellow-tan Yellow0.1% Tyrosine S Colorless Colorless Colorless Gray-green Gray-green R Red Red Red Red RedCasein starch S Gray-green Gray-green Gray-green Gray-green Gray-green R Pale gray-green Pale gray-green Pale gray-green Pale gray-green Pale gray- green__________________________________________________________________________ S = Surface R = Reverse *Dietz, A. 1954. Ektachrome transparencies as aids in actinomycete classification. Ann. N.Y. Acad. Sci. 60: 152-154.
TABLE 2______________________________________Reference Color Characteristics of Streptomyces espinosusCultures______________________________________ De- ter- Color mi- Harmony Manual, 3rd ed., 1948*Agar na- NRRL NRRL NRRL NRRL NRRLMedium tion 3890 5729 5730 5731 11081______________________________________Bennett's S a a a 241/2fe 241/2ih 241/2fe 241/2ih 241/2ih 11/2ge 1ge R 11/2ca 11/2ca 11/2gc 2ec 2ea 2fb 2fb 11/2ec 2ic 2ec 2ec P -- -- -- -- --Czapek's S 1 li 1 li 2ig 2ig 2igsucrose 2ig 2ig R 1ec 1ec 2fe 1ec 2fe 2ig 2ig P -- -- -- -- 'Maltose- S 1ig 241/2fe 3ba 241/2fe 241/2ihtryptone 11/2ge 1ih 241/2ih 1ih R 2ge 2gc 2ic 11/2ie 2ie 2ec 11/2ge 2gc 11/2ge P -- -- -- -- --Hickey- S 1ig 1ig 3ba 241/2ih 241/2ihTresner 241/2ih(modified) R 2gc 2ec 2gc 2gc 2ec P -- -- -- -- --Yeast extract- S 241/2ih 241/2ih 241/2ih 241/2ih 241/2ihmalt extract 2ih 2ih(ISP-2) R 2le 2gc 2le 2gc 2le 2gc 2ie P -- -- -- -- --Oatmeal S 1ig 1ig 241/2fe 241/2ih 241/2fe(ISP-3) 11/2ig 2fe R 1ec 1ec 2ec 2ec 2ec 11/2ge 2ec P -- -- -- -- --Inorganic-salts S 241/2ih 241/2ih 241/2ih 241/2ih 241/2ihstarch (ISP-4) 3ih 1ih R 2ca 2gc 2gc 2ec 2gc 2gc P 2ec 2ec 2ec 2ig 2ecGlycerol- S 1ig a 3ba 11/2ig 241/2ihasparagine 2ih 2fe 241/2fe(ISP-5) R 1ec 2gc 2ge 2ge 2ig 2ge 2ge P 2ge -- -- -- --______________________________________ De- ter- mi- NBS Circular 553, 1955**Agar Na NRRL NRRL NRRL NRRL NRRLMedium tion 3890 5729 5730 5731 11081______________________________________Bennett's S 263gm 263gm 263gm 122gm 122m 122gm 122m 122m 127g 109gm 127g 127g 109gm R 89gm 89gm 102g 90gm 86gm 87g 87g 105gm 90gm 87gm 89m 89m 93m 90gm 90gm P -- -- -- -- --Czapek's S 110gm 110gm 110g 110g 110gsucrose 110g 112m 112m 112m 112m R 112m 112m 94g 121m 94g 121m 121m 112gm 122g 112gm 122g 122g 110g 110g P 112m 112m -- -- --Maltose- S 109gm 122gm -- 122gm 122mtryptone 110g 112m 122m 127g 109gm 113g 127g 112m 113g R 90gm 90gm 87gm 106gm 91gm 90gm 109gm 94g 90gm 106g 109gm P -- -- -- -- --Hickey- S 109gm 109gm -- 122m 122mTresner 110g 110g 127g 127g 127g(modified) R 90gm 90gm 90gm 90gm 90gm P -- -- -- -- --Yeast extract- S 122m 122m 122m 122m 122mmalt extract 127g 127g 127g 127g 127g(ISP-2) 122m 122m 113g 113g R 88gm 90gm 88gm 90gm 88gm 94g 91gm 94g 94g 90gm 94g P -- -- -- -- --Oatmeal S 109gm 109gm 122gm 122gm 122gm(ISP-3) 110g 110g 94g 112gm R 121m 121m 90gm 90gm 90gm 109gm gm P -- -- -- -- --Inorganic-salts S 122m 122m 122m 122m 122mstarch (ISP-4) 113g 112m 265m 113g R 89gm 90gm 90gm 90gm 90gm 90gm P 90gm 90gm 90gm 110g 90gm 112mGlycerol- S 109gm 263gm -- -- 122masparagine 110g 94g 122gm 127g(ISP-5) 112m 112gm 113g R 121m 90gm 94m 94m 110g 122g 94gm 109gm 109gm 112m 94m 109gm 109gm P -- -- -- -- --______________________________________ S = Surface R = Reverse P = Pigment m = matte g = glossy gm = glossy or matte surface of chip *Jacobson, E., W.C. Granville, and C.E. Foss. 1948. Color harmony manual, 3rd ed. Container Corporation of America, Chicago, Illinois. **Kelly, K.L., and D.B. Judd. 1955. The ISCCNBS method of designating colors and a dictionary of color names. U.S. Dept. Comm. Circ. 553.
TABLE 3______________________________________ Color Code for TABLE 2Color Harmony Manual, NBS Circular 553,3rd ed., 1948* 1955**Color Colorchip Color name chip Color name______________________________________a White 263gm White1ec Light citron gray, putty 121m Pale yellow green 122g Grayish yellow green1ge Citron gray 109gm Light grayish olive1ig Olive gray 109gm Light grayish olive 110g Grayish olive1ih Olive gray 112m Light olive gray 113g Olive gray 127g Grayish olive green1 li Light olive drab 110gm Grayish olive11/2 ca Cream 89gm Pale yellow11/2ec Biscuit, ecru, oatmeal, 90gm Grayish yellow sand 93m Yellowish gray11/2 gc Dusty yellow 102g Moderate greenish yellow 105gm Grayish greenish yellow11/2 ge Light olive gray 109gm Light grayish olive11/2 ie Light olive 106gm Light olive11/2 ig Olive gray -- --2ca Light ivory eggshell 89gm Pale yellow2ea Light wheat, light maize 86gm Light yellow2ec Biscuit, ecru, oatmeal, 90gm Grayish yellow sand2fb Bamboo, buff, straw, 87g Moderate yellow wheat 89m Pale yellow2fe Covert gray 94g Light olive brown 122gm Light olive gray2gc Bamboo, chamois 90gm Grayish yellow2ge Covert tan, griege 94m Light olive brown 109gm Light grayish olive2ic Honey gold, light gold 87gm Moderate yellow2ie Light mustard tan 91gm Dark grayish yellow 94g Light olive brown 106g Light olive2ig Slate tan 110g Grayish olive 112m Light olive gray2ih Dark covert gray 112m Light olive gray 113g Olive gray2le Mustard, old gold 88gm Dark yellow 94g Light olive brown3ba Pearl, shell tint -- --3ih Beige gray, mouse 113g Olive gray 265m Medium gray241/2 fe Light mistletoe gray 122gm Grayish yellow green241/2 ih Mistletoe gray 122m Grayish yellow green 127g Grayish olive green______________________________________ *Jacobson, E., W.C. Granville, and C.E. Foss. 1948. Color harmony manual, 3rd ed. Container Corporation of America, Chicago, Illinois. **Kelly, K.L., and D.B. Judd. 1955. The ISCCNBS method of designating colors and a dictionary of color names. U.S. Dept. Comm. Circ. 553.
TABLE 4__________________________________________________________________________Cultural and Biochemical Characteristics of Streptomyces espinosusCultures De- ter- mi- na- NRRL NRRL NRRL NRRL NRRLMedium tion 3890 5729 5730 5731 11081__________________________________________________________________________AgarPeptone-iron S Gray-white to Gray-white to Trace gray- Gray-green- Gray-green- gray-green gray-green green-white white white R Yellow Yellow Yellow Yellow Yellow O Melanin negative Melanin negative Melanin negative Melanin negative Melanin negativeCalcium S Fair to good Trace gray- Trace gray- Trace gray- Trace gray-malate gray-green green green green green R Pale gray Pale gray Pale gray Pale gray Pale gray O Malate not Malate not Malate not Malate not Malate not solubilized solubilized solubilized solubilized solubilizedGlucose- S Gray-white to Gray-white to Fair gray- Fair gray- Fair gray-asparagine gray-yellow trace green white white white R Light yellow Cream Cream Cream Cream to creamSkim milk S White on edge to White on edge White on edge White on edge White on edge light gray- to gray-green- green yellow pink R Deep yellow to Yellow-tan Yellow-tan Yellow-tan Yellow-tan yellow-tan O Yellow to yellow- Yellow-tan Yellow-tan Yellow-tan Yellow-tan tan pigment pigment pigment pigment pigment Casein solu- Casein solu- Casein solu- Casein solu- Casein solu- bilized under bilized under bilized under bilized under bilized under growth to growth to growth growth growth completely completelyTyrosine S Good to heavy Good to heavy Good gray-green Good gray-green Good gray-green gray-green gray-green R Red-tan to Red-tan to Red-tan Red-tan Red-tan red-brown red-brown O Red-tan to red- Red-tan to red- Red-tan pigment Red-tan pigment Red-tan pigment brown pigment brown pigment Tyrosine solu- Tyrosine solu- Tyrosine solu- Tyrosine solu- Tyrosine solu- bilized bilized bilized bilized bilizedXanthine S Good gray-green Good gray-green Good gray-green Good gray-green Good gray-green to good only on periphery R Pale yellow Pale yellow- Cream Cream Cream to cream green to cream O Xanthine not Xanthine not Xanthine not Xanthine not Xanthine not solubilized solubilized solubilized solubilized solubilizedNutrient S Good gray-green Good gray-green Good gray-green Good gray-green Good gray-greenstarch R Cream-olive Cream-olive Cream-olive Cream-olive Cream-olive O Starch Starch Starch Starch Starch hydrolyzed hydrolyzed hydrolyzed hydrolyzed hydrolyzedYeast extract- S Good to heavy Good gray-green Good gray-green Good gray-green Good gray-greenmalt extract gray-green R Pale yellow-tan Cream-yellow-tan Cream-yellow-tan Cream-yellow-tan Cream-yellow-tan to cream- yellow-tanBennett's S Cream to heavy Gray-green-white Trace gray-cream Cream Gray-green-white gray-green to heavy gray- green R Yellow to olive Cream to olive Yellow Yellow Yellow-orangeCzapek's S Gray-green Gray-green Gray-green Gray-green Gray-greensucrose R Gray-green Gray-green Gray-green Gray-green Gray-greenMaltose- S Heavy gray-green Good to heavy Gray-green- Pale gray-green Gray-greentryptone gray-green white white R Olive to orange- Olive to cream Yellow Yellow-olive Yellow-orange yellowHickey-Tresner S Heavy gray-green Heavy gray-green Gray-green-white Gray-green Heavy gray-green(modified) R Olive-orange Cream Deep yellow-tan Yellow-olive Olive-orangePeptone-yeast S Cream to pale Pale pink to Cream to pale Cream with trace Cream to paleextract-iron pink gray-green pink gray-green to pink(ISP-6) pale pink O Melanin negative Melanin negative Melanin negative Melanin negative Melanin negativeTyrosine S Gray-green-white Gray-green-white Gray-green Gray-green Gray-green(ISP-7) to gray-green to gray-green R Pale yellow- Pale yellow- Gray-cream Gray-cream Gray-green green to tan green to gray- cream O Melanin-negative Melanin-negative Melanin-negative Melanin-negative Melanin-negativeGelatinPlain O Colorless vege- Colorless vege- Gray-white Gray-white Gray-green-white tative growth tative growth aerial growth; aerial growth; aerial growth; to gray-white to gray-white liquefaction liquefaction liquefaction aerial growth; aerial growth; complete complete complete liquefaction liquefaction 1/2 to 1/2 to complete completeNutrient O White to gray- White to gray- Gray white Trace gray-white Gray-white white aerial white aerial aerial growth; aerial growth; aerial growth; growth; growth; liquefaction liquefaction liquefaction liquefaction liquefaction complete complete complete 1/3 to 1/3 to complete completeBrothSynthetic O White to pink- Trace white to Trace white Trace white Trace whitenitrate cream aerial pink-cream aerial growth aerial growth aerial growth growth on sur- aerial growth on surface on surface on surface face pellicle; on surface pellicle; pellicle; pellicle; growth through- pellicle; growth through- trace growth growth through- out medium; growth through- out medium; throughout out medium; nitrate not out medium; nitrate not medium; nitrate nitrate not reduced to nitrate not reduced to not reduced to reduced to nitrite reduced to nitrite nitrite nitrite nitriteNutrient O Gray-green-white Gray-green-white Gray-green-white Trace white Gray-green-whitenitrate aerial growth aerial growth aerial growth aerial growth aerial growth on surface on surface on surface on surface on surface pellicle; pellicle; pellicle; pellicle; pellicle; flocculent flocculent flocculent compact to flocculent bottom growth; bottom growth; bottom growth; flocculent bottom growth; nitrate test: nitrate not nitrate not bottom growth; nitrate not neither reduced to reduced to nitrate not reduced to nitrate nor nitrate nitrite reduced to nitrite nitrite nitrite present or nitrate not reduced to nitriteLitmus milk O Cream to green Pink-tan to Tan aerial Green-white Tan aerial aerial growth gray-green growth on aerial growth growth on on yellow to aerial growth surface ring; on surface surface ring; tan surface on surface litmus reduced; ring; partial partial ring; litmus ring; litmus peptonization reduction; reduction; reduced; reduced; pH 7.3 partial partial peptonization; peptonization; peptonization; peptonization; pH 7.3 pH 7.3 pH 7.1 pH 7.4__________________________________________________________________________ S = Surface R = Reverse P = Pigment O = Other Characteristics
TABLE 5______________________________________Utilization Of Carbon Compounds ByStreptomyces espinosus Cultures In The SyntheticMedium Of Pridham And Gottlieb* NRRL NRRL NRRL NRRL NRRL 3890 5729 5730 5731 11081______________________________________ CONTROL (+)** (-)** (+) (+) (+) 1. D-Xylose + + + + + 2. L-Arabinose + + + + + 3. Rhamnose + + + + + 4. D-Fructose + (+),+ + + + 5. D-Galactose + + + + + 6. D-Glucose + + + + + 7. D-Mannose + + + + + 8. Maltose + + + + + 9. Sucrose (+) (-),(+) (+) (+) (+)10. Lactose + + + + +11. Cellobiose + + + + +12. Raffinose (+) (-) (+) (+) (+)13. Dextrin + + + + +14. Inulin (+),(-) (-) (+) (+) (+)15. Soluble starch + + + + +16. Glycerol + + + + +17. Ducitol (+),(-) (-) (+) (+) (+)18. D-Mannitol + + + + +19. D-Sorbitol + ,(-) (-) + + +20. Inositol + (+) + + +21. Salicin (+) (+) + (+) (+)22. Phenol (+),(-) (-) (+) (+) (+)23. Cresol - - - - -24. Na Formate (+),- (-)- (+) (+) (+) CONTROL (+)** (-)** (+) (+) (+)25. Na Oxalate (+),(-) (+),(-) (+) (+) (+)26. Na Tartrate (+),(-) (-),- (+) (+) (+)27. Na Salicylate - - - - -28. Na Acetate (+) (+) (+) (+) (+)29. Na Citrate + (+) + (+) +30. Na Succinate (+),(-) (-) (+) (+) (+)______________________________________ + = Good utilization (+) = Poor utilization (-) = Doubtful utilization - = No growth *Pridham, T.G., and D. Gottlieb. 1948. The utilization of carbon compound by some Actinomycetales as an aid for species determination. J. Bacteriol 56: 107-114. **Results from different studies
TABLE 6______________________________________Utilization Of Carbon Compounds ByStreptomyces espinosus Cultures In The SyntheticMedium Of Shirling And Gottlieb* NRRL NRRL NRRL NRRL NRRL 3890 5729 5730 5731 11081______________________________________CONTROLNegative-basal .+-.** -,.+-.** .+-. .+-. .+-.mediumPositive-basal + + + + +medium plusD-glucoseCarbon compoundsL-Arabinose .+-.,++ .+-.,+ .+-. .+-. .+-.Sucrose - .+-.,- - - -D-Xylose ++ .+-.,++ ++ ++ ++Inositol ++ .+-.,+ ++ ++ ++D-Mannitol ++ ++ ++ ++ ++D-Fructose ++ .+-.,+ ++ ++ ++Rhamnose ++ ++ ++ ++ ++Raffinose - - - - -Cellulose +,++ .+-.,- + + +______________________________________ ++ Strong utilization .+-. Utilization doubtful + Positive - Utilization negative *Shirling, E.B., and D. Gottlieb. 1966. Methods for characterization of Streptomyces species. Int. J. Syst. Bacteriol. 16: 313-340. **Results from different studies
The new compound of the invention is produced when the elaborating organism is grown in an aqueous nutrient medium under submerged aerobic conditions. It is to be understood, also, that for the preparation of limited amounts surface cultures and bottles can be employed. The organism is grown in a nutrient medium containing a carbon source, for example, an assimilable carbohydrate, and a nitrogen source, for example, an assimilable nitrogen compound or proteinaceous material. Preferred carbon sources include glucose, brown sugar, sucrose, glycerol, starch, cornstarch, lactose, dextrin, molasses, and the like. Preferred nitrogen sources include cornsteep liquor, yeast, autolyzed brewer's yeast with milk solids, soybean meal, cottonseed meal, cornmeal, milk solids, pancreatic digest of casein, fish meal, distillers' solids, animal peptone liquors, meat and bone scraps, and the like. Combinations of these carbon and nitrogen sources can be used advantageously. Trace metals, for example, zinc, magnesium, manganese, cobalt, iron, and the like, need not be added to the fermentation medium since tap water and unpurified ingredients are used as components of the medium prior to sterilization of the medium.
Production of the compound of the invention can be effected at any temperature conductive to satisfactory growth of the microorganism, for example, between about 18.degree. and 40.degree. C., and preferably between about 20.degree. and 28.degree. C. Ordinarily, optimum production of the compound is obtained in about 3 to 15 days. The medium normally remains neutral during the fermentation. The final pH is dependent, in part, on the buffers present, if any, and in part on the initial pH of the culture medium.
When growth is carried out in large vessels and tanks, it is preferable to use the vegetative form, rather than the spore form, of the microorganism for inoculation to avoid a pronounced lag in the production of the new compound and the attendant inefficient utilization of the equipment. Accordingly, it is desirable to produce a vegetative inoculum in a nutrient broth culture by inoculating this broth culture with an aliquot from a soil stock, an agar plug stored above liquid N.sub.2, or a slant culture. When a young, active vegetative inoculum has thus been secured, it is transferred aseptically to large vessels or tanks. The medium in which the vegetative inoculum is produced can be the same as, or different from, that utilized for the production of the new compound, so long as good growth of the microorganism is obtained.
A variety of procedures can be employed in the isolation and purification of the compound of the subject invention, for example, solvent extraction, partition chromatography, silica gel chromatography, liquid-liquid distribution in a Craig apparatus, adsorption on resins, and crystallization from solvents.
In a preferred recovery process the compound of the subject invention is recovered from the culture medium by separation of the mycelia and undissolved solids by conventional means, such as by filtration or centrifugation. The antibiotic is recovered from the filtered or centrifuged broth by extraction with a suitable solvent for acanthomycin, for example, water-immiscible alcohols like 1-butanol (preferred). The solvent extract containing the desired antibiotic is concentrated and then subjected to purification procedures as disclosed infra.
Before disclosing purification procedures, there is first disclosed alternate recovery procedures. Acanthomycin can be recovered from the fermentation whole beer by filtration followed by adsorption of the antibiotic on a non-ionic resin. Suitable non-ionic resins include resins comprising a non-ionic macro porous copolymer of styrene crosslinked with divinylbenzene. Non-ionic resins of this type, marketed under the trade names Amberlite XAD-2 and XAD-4, are disclosed in U.S. Pat. No. 3,515,717.
Further, activated carbon, alumina and related adsorbants can be used to recover acanthomycin from filtered fermentation beer.
Since acanthomycin is an acidic compound, anion exchangers, for example, Dowex-1; Dowex-2; Dowex 21K (Dow Chemical Co., USA) or Amberlite IRA-900; -IRA-904; -IR-45 (Rohm and Haas Co., USA) can be used to recover the antibiotic from filtered fermentation beer. The antibiotic is adsorbed onto the resin and then eluted by the use of an aqueous solution of inorganic acids, salts like ammonium chloride and bases like ammonium hydroxide, dilute sodium hydroxide, and the like.
After recovery of acanthomycin from the fermentation beer, the recovery preparation is then subjected to purification procedures which will ultimately yield a purified crystalline preparation of acanthomycin. It should be recognized, however, that any of the crude preparations containing acanthomycin, including the fermentation beer, can be used for purposes well known to those skilled in the antibiotic art. For example, such crude preparations can be used as feed for animals. Where relatively pure preparations of acanthomycin are desired, the crude preparaions of acanthomycin can be purified by the following procedures, or obvious equivalents thereof.
The crude preparation of acanthomycin is first mixed with a suitable solvent, for example, ethyl acetate, and the like, to precipitate a material containing acanthomycin. This material is isolated by filtration and then distributed between water and a suitable solvent for acanthomycin, for example, 1-butanol. The solvent phase containing acanthomycin, is separated out, then mixed with water. The pH of the mixture is adjusted to a basic pH, for example, about 7.9, with concentrated base, for example, ammonium hydroxide. The resulting solvent and aqueous phases are separated and then the aqueous phase is extracted with a solvent for acanthomycin. The solvent extract containing acanthomycin is then subjected to chromatographic procedures on a suitable resin, for example, Amberlite IRA-904 anion exchanger in the chloride form. This resin is a product of Rohm and Haas Company, USA. The chromatographic resin column is eluted with aqueous ammonium chloride.
Active fractions from the above chromatographic procedure are then subjected to another chromatographic procedure to separate acanthomycin from ammonium chloride. The resin used in this procedure is a non-ionic resin known as Amberlite XAD-7, again a product of Rohm and Haas Co. This resin is eluted with a methanol-water mixture to give fractions containing acanthomycin. The active fractions are combined and then concentrated to an aqueous and freeze-dried to give a relatively pure preparation of acanthomycin.
A highly purified preparation of acanthomycin can be obtained by subjecting the above-described freeze-dried preparation to chromatography using DEAE-Sephadex (A-25) supplied by Pharmacia Fine Chem., Uppsala, Sweden. The preparation containing acanthomycin is dissolved in ammonium chloride and this solution is then passed over a column containing the DEAE-Sephadex (A-25). Fractions active against B. subtilis and S. pyogenes are pooled. Acanthomycin is isolated from the resulting three pools by use of chromatographic procedures using a non-ionic resin, for example, Amberlite XAD-7.
In processing the pools over the Amberlite XAD-7 resin, the resin is eluted with a methanol-water mixture to give acanthomycin. The ammonium salt of acanthomycin is made by adjusting the pH of these fractions to about 7.5 with ammonium hydroxide.
Acanthomycin is active against S. aureus and can be used to disinfect washed and stacked food utensils contaminated with these bacteria; it can also be used as a disinfectant on various dental and medical equipment contaminated with S. aureus. Further, acanthomycin and its salts can be used as a bacteriostatic rinse for laundered clothes, and for impregnating papers and fabrics; and, they are also useful for suppressing the growth of sensitive organisms in plate assays and other microbiological media. Since acanthomycin is active against B. subtilis, it can be used in petroleum product storage to control this microorganism which is a known slime and corrosion producer in petroleum products storage.
It is to be understood that the microbiological process disclosed herein, though described in detail with reference to Streptomyces espinosus subsp. acanthus, NRRL11081, is not limited to this particular microorganism deposit. It is intended that any microorganism meeting the cultural characteristics disclosed herein, or substantial equivalents thereof, wherever deposited in the world, is a part of the subject microbiological process. Further, it is intended that this invention include strains or mutants of the said microorganism which can be produced by procedures well known in the art, for example, by subjecting the novel microorganism to x-ray or ultraviolet radiation, nitrogen mustard, phage exposure, and the like.
Since acanthomycin is an acidic compound, it forms salts with both inorganic and organic bases. For example, the sodium, potassium, ammonium, calcium, and the like, salts can be made by neutralizing a solution of acanthomycin in aqueous methanol. Salts of acanthomycin with organic bases (primary, secondary and tertiary amines) for example, pyridine, piperidine, and the like, can be made by mixing of equal molar amounts of acanthomycin and the desired organic base.

The following examples are illustrative of the process and products of the subject invention but are not to be construed as limiting. All percentages are by weight and all solvent mixture proportions are by volume unless otherwise noted.
EXAMPLE 1
Part A. Fermentation
An agar slant of Streptomyces espinosus subsp. acanthus, NRRL 11081, is used to inoculate a series of 500-ml Erlenmeyer flasks, each containing 100 ml of sterile preseed medium consisting of the following ingredients:
______________________________________Glucose monohydrate 25 g/literPharmamedia* 25 g/literTap water q.s. 1 liter______________________________________ *Pharmamedia is an industrial grade of cottonseed flour produced by Traders Oil Mill Company, Fort Worth, Texas.
The preseed medium presterilization pH is 7.2. The preseed inoculum is grown for three days at 28.degree. C. on a Gump rotary shaker operating at 250 r.p.m. and having a 21/2 inch stroke.
Preseed inoculum (300 ml), prepared as described above, is used to inoculate a seed tank containing 20 liters of sterile seed medium consisting of the following ingredients:
______________________________________Glucose monohydrate 25 g/literPharmamedia 25 g/literTap water Balance______________________________________
The inoculated seed medium is incubated at a temperature of 28.degree. C. for 2 days while being agitated at a rate of 400 r.p.m. and aerated at a rate of 10 standard liters per minute with a back pressure of 10 psig.
After 2 days incubation, the seed medium is used to inoculate (the inoculation rate is 5 liters of seed inoculum per 100 liters of fermentation medium) a 250 liter fermentation tank containing sterile fermentation medium consisting of the following ingredients:
______________________________________B and F Distillers Solubles* 40 g/literNaCl 5 g/literNaNO.sub.3 10 g/literUcon** 2 ml/literTap water q.s. BalancepH - 7.0 (presterilization)______________________________________ *Distillers residue supplied by BrownForman Distillers Corp., P.O. Box 1080, Louisville, KY 40201. **a synthetic defoamer supplied by Union Carbide, N.Y., N.Y.
The fermentation tank is incubated at a temperature of 28.degree. C., with agitation of 200 r.p.m. and aeration at 150 standard liters per minute with back pressure at 10 psig. Harvest is usually after 3 to 12 days of fermentation.
The fermentation can be monitored by a disc-plate assay using B. subtilis as the test organism and deionized H.sub.2 O as diluent. A unit volume (0.08 ml) of solution containing the substance to be assayed is placed on a 12.7 mm paper disc which is then placed on an agar plate seeded with the assay organism. The agar plate is then incubated for 16 to 18 hours at 32.degree. to 37.degree. C. A biounit (BU) is defined as the concentration of the antibiotic which gives a 20 mm zone of inhibition under the standard assay conditions. Thus, if for example a fermentation beer has to be diluted 1/100 to give a 20 mm zone of inhibition, the potency of such beer is 100 BU/ml.
Part B. Recovery
Whole fermentation beer (ca. 5,000 l), obtained as described above, is adjusted to pH 2.75 with 6 N aqueous hydrochloric acid. The adjusted whole beer is then filtered using filter aid (diatomaceous earth). The mycelial cake is washed with 1,000 l of water. The washed filter cake is discarded. The aqueous wash is combined with the clear filtrate and the resulting solution (5,750 l) is extracted with 1,600 l of 1-butanol. The spent beer is discarded. The butanolic extract is concentrated to a volume of ca. 8 l (4.5 kg). This material is designated preparation ADA-117A.
Part C. Purification
(No. 1) Precipitation
One liter of preparation ADA-117A, isolated as described in the recovery section is poured into 10 l of ethyl acetate under stirring. The mixture is stirred at room temperature for one hour. The precipitated material containing acanthomycin is isolated by filtration and treated as described, infra under distribution.
(No. 2) Distribution
The precipitate is distributed between 2.5 l of water and 2.5 l of 1-butanol. The butanol phase (ADA-31C) is mixed with 7 l of water. The pH of the mixture is adjusted to 7.9 with concentrated ammonium hydroxide. The two phases are separated and designated Butanol-1 and Aqueous-1. Aqueous-1 is then extracted 3 times with 2.5 l portions of 1-butanol each time at pH 7.9 (adjusted with ammonium hydroxide). The three butanolic extracts (designated Butanol-2, Butanol-3-and Butanol-4) are combined with Butanol-1 and the combined extract is kept as ADA-31D.
Testing for bioactivities showed the following:
______________________________________ Zone (mm) S. B. S. B. S.Preparation pyogenes cereus aureus subtilis lutea______________________________________ADA-31C 36 18 20 23 27ADA-31D 36 16 24 26.5 27______________________________________
Preparation ADA-31D containing most of the bioactivity was treated as described below.
(No. 3) Amberlite IRA-904 Chromatography
The column is prepared from 2 liters of Amberlite IRA-904 anion exchanger in the chloride form. (Product of Rohm and Haas, USA).
The butanolic extract (ADA-31D), obtained above, is mixed with one half of its volume of methanol and one half of its volume of water and this solution is passed through the column at a rate of 50-70 ml/minute. The spent is collected in three equal fractions designated "Spent-1," "Spent-2" and "Spent-3" which are found bioinactive.
The column is washed with 6 l of water. This eluate, kept as "Aqueous," is found bioinactive.
The column is then eluted with 2% aqueous ammonium chloride. Fractions of 20 ml are collected and combined in 500 ml fractions and tested. Results follow. Zone size in mm.
______________________________________ S. B. S. B. S.Fraction No. pyogenes cereus aureus subtilis lutea______________________________________1 0 0 0 0 02 0 0 0 0 03 35 17 25 26.5 254 39 21 27.5 30 295 40 22 28.5 31 306 39 21 28 29.5 29.57 39 20.5 27.5 29.5 28.58 38 20 27 29 28.59 37 20 27 28.5 28.510 -- 18 25 28 2811 -- 16.5 24 27.5 27.512 -- 16 23 26.5 26.513 -- 15.5 23 25.5 2514 -- 15.5 22.5 24.5 24.515 -- traces 21 22.5 2316 -- 0 18 18 1717 -- 0 0 0 018 -- -- -- -- --______________________________________
Fractions 3-18 are combined, adjusted to pH 5.5 with 6 N aqueous hydrochloric acid and this solution is used as starting material for the chromatography described below.
(No. 4) Amberlite XAD-7 Chromatography
Separation of Acanthomycin from Ammonium Chloride
The column is prepared from 2 l of Amberlite XAD-7 (Rohm and Haas Co., USA).
The solution described above containing acanthomycin in mixture with ammonium chloride (8 l, pH 5.5) is passed over the column at a rate of 80-100 ml/minute. The spent is collected in two equal fractions designated "Spent-1" and "Spent-2".
The column is washed with 4 l of water (flow rate 80-100 ml/minute). Four equal fractions (Aqueous-1; -2; -3; -4) are collected.
The column is then eluted with methanol-water (70:30 v/v). Fractions of 20 ml are collected. Testing shows the following. Zone size in mm.
______________________________________ S. S. B. B. S.Fraction pyrogenes aureus cereus subtilis lutea______________________________________Start-Material 35 24.5 19 27.5 26Spent-1 0 0 0 0 0Spent-2 0 0 0 0 0Aqueous-1 0 0 0 0 0Aqueous-2 0 0 0 0 0Aqueous-3 0 0 0 0 0Aqueous-4 0 0 0 0 0MeOH-Water 10 0 0 0 0 0 20 0 0 0 0 0. . . . . . 80 0 0 0 0 0 90 42 29 23.5 35.5 37100 40 28.5 24 33 35110 30 19.5 traces 22.5 24120 24 traces 0 17.5 17130 21.5 0 0 traces traces140 18 0 0 0 0150 15.5 0 0 0 0160 16 0 0 0 0170 15 0 0 0 0180 traces 0 0 0 0190 0 0 0 0 0200 0 0 0 0 0. . . . . .250 0 0 0 0 0______________________________________
Fractions 85-125 are combined. The solution is concentrated to an aqueous and freeze-dried to give preparation ADA-34.1, 3.7 g containing acanthomycin.
Using 5.5 l of preparation ADA-117A, obtained as described in Part B., supra, as the starting material, and using the above-described purification procedure (precipitation, distribution, IRA-904 and XAD-7 chromatographies) a total of 21.71 g of material of highly active acanthomycin is obtained (Prep. ADA-113.4).
Highly purified acanthomycin, obtained as described above, is purified by DEAE-Sephadex chromatography as shown below.
(No. 5) DEAE-Sephadex Chromatography
Eight hundred g of DEAE-Sephadex (A-25) (Pharmacia Fine Chem., Uppsala, Sweden) is slurried in 0.05 N ammonium chloride solution adjusted to pH 8.5 with ammonium hydroxide. The mixture is allowed to stand at room temperature for 12 hours. The swelled resin is then poured into a column. The column is then washed with 25 l of solvent.
The starting preparation, ADA-113.4 (20 g), obtained as described above, is dissolved in 200 ml of the 0.05 N ammonium chloride (pH 8.5) solvent and this solution is added on the top of the column. The column is then eluted with the same solvent. Fractions are tested for bioactivity against B. subtilis and S. pyogenes. Results follow.
______________________________________ Zone (mm)Fraction No. Volume (1) B. subtilis S. pyogenes______________________________________1 2.0 0 02 2.0 0 03 8.5 0 04 12.0 0 05 4.0 0 06 4.0 0 07 3.0 0 08 12.0 0 09 4.0 15 2410 3.5 17 2811 9.0 20 2812 7.5 21 28.513 8.5 21 3014 6.0 23 30.515 9.0 22.5 31.516 4.0 22.5 31.517 3.0 21 30.518 12.0 20.5 30.519 4.0 20.5 3020 4.0 20 3021 12.0 20 3022 9.0 20 3123 3.0 21 3124 12.0 19 29.525 4.0 19 29.526 4.0 18 29.527 9.0 19 2928 12.0 -- --29 4.0 18 2830 4.0 18 27.531 12.0 17 2832 12.0 17 2733 12.0 17.5 2634 12.0 17 2635 10.0 17 2536 12.0 17 2537 7.0 15 2438 12.0 15 23.539 12.0 traces 23.540 12.0 0 2441 7.0 0 2542 12.0 0 --43 12.0 0 --______________________________________
The following pools are made:
Pool I: Fractions 9-12 ca. 24 l
Pool II: Fractions 13-20 ca. 50.5 l
Pool III: Fractions 21-43 ca. 214 l
Acanthomycin is isolated from the above pools by Amberlite XAD-7 chromatographies as described below.
(A) Isolation of Acanthomycin from Pool I
Amberlite XAD-7 Chromatography
The column is prepared from 1 l of Amberlite XAD-7. Pool I is adjusted to pH 3.0 with 6 N aqueous hydrochloric acid and passed over the column with a flow rate of 25 ml/minute. The spent is found bioinactive and is discarded. The column is then washed with 3 l of water. The aqueous wash is found bioinactive and is also discarded. The column is then eluted with methanol-water (70:30 v/v). Fractions of 20 ml are collected. Testing shows the following.
______________________________________ Zone (mm)Fraction No. B. subtilis S. pyogenes______________________________________10 0 020 0 030 0 040 0 050 20.5 23.560 25 29.570 26 32.5 80 27 34 90 26.5 34.5100 26 34.5110 25.5 33.5120 25.5 32130 24 31140 23 31150 21.5 28.5160 20 27.5170 18 24.5180 16 22190 0 18200 0 16210 0 0. . .400 0 0______________________________________
Fractions 46-200 are combined, adjusted to pH 7.5 with ammonium hydroxide, concentrated to an aqueous solution and freeze-dried to give preparation ADA-44.1, 550 mg (acanthomycin ammonium salt).
(B) Isolation of Acanthomycin from Pool II
Amberlite XAD-7 Chromatography
The column is prepared from 2 l of Amberlite XAD-7. Pool II is adjusted to pH 3.0 with 6 N aqueous hydrochloric acid. A precipitate is formed which is separated by filtration and dried (ADA-45). This material yields crystalline acanthomycin, as described infra. The filtrate is passed over the column at a rate of 50 ml/minute. The spent solution is found bioinactive and is discarded. The column is then washed with 6 l of water. The aqueous wash is found bioinactive and is also discarded. The column is then washed with 6 l of water. The aqueous wash is found bioinactive and is also discarded. The column is then eluted with methanol-water (70:30 v/v). Fractions (20 ml) are collected. Testing shows the following.
______________________________________ Zone(mm)Fraction No. B. subtilis S. pyogenes______________________________________ 10 0 0 20 0 0. . .140 0 traces150 0 15160 0 16170 0 16180 0 18190 16 22200 19 25210 22 29220 24 33.5230 27 33.5240 27.5 36250 28.5 38260 28 37.5270 28 38.5280 27 34.5290 27 33.5300 26 33310 25.5 32320 24 31330 23.5 29340 23 29.5350 22 29360 21 29370 20.5 27.5380 21 28390 20 27400 19.5 26410 19.5 26420 19 25430 19 24440 18 23450 17 23.5460 17 23470 17 23480 16 22______________________________________
The column is then eluted with methanol-0.05 N ammonium hydroxide. Fractions of 20 ml are collected. Results follow:
______________________________________ Zone (mm)Fraction No. B. subtilis S. pyogenes______________________________________10 15.5 21.520 15 2130 15 2140 16 20.550 16 19.560 15 17.570 15 1980 15 20.590 17 23.5100 20 27.5110 25 33120 26 34.5130 20 25.5140 0 traces______________________________________
The following combinations are made:
I: Fractions (Methanol-water 70:30); 190-219
II: Fractions (Methanol-water 70:30); 220-399
III: Fractions (Methanol-water 70:30); 440-483
IV: Fractions (Methanol-0.05 NH.sub.4 OH 70:30); 100-140
The combined solutions are concentrated to dryness to give the following:
I: Acanthomycin, prep. ADA-46.3; 70 mg
II: Acanthomycin, prep. ADA-46.4; 450 mg
III: Acanthomycin, prep. ADA-46.5; 50 mg
IV: Acanthomycin ammonium salt, prep. ADA-46.6; 250 mg
As mentioned above, a precipitate, formed by acidification of pool II, is isolated by filtration and kept as ADA-45. Trituration of this material with methanol, followed by concentration of the methanolic solution to dryness, yields a crystalline residue. Trituration of the residue with ether affords colorless crystalline acanthomycin which is isolated by filtration and dried (Prep. ADA-46.1, 420 mg).
(C) Isolation of Acanthomycin from Pool III
Amberlite XAD-7 Chromatography
The column is prepared from 3 l of Amberlite XAD-7.
Starting material, Pool III, prepared as described above, is adjusted to pH 3.0 with 6 N aqueous hydrochloric acid and this solution is passed over the column at a flow rate of 75 ml/minute. The spent is found bioinactive and is discarded. The column is washed with 9 l of water. The aqueous wash is found bioinactive and is discarded. The column is eluted with methanol-water (70:30 v/v). Fractions (20 ml) are collected. Testing results follow.
______________________________________ Zone (mm)Fraction No. B. subtilis S. pyogenes______________________________________ 10 0 0 20 0 0. . .120 0 0130 0 18140 0 20150 traces 23160 16 27170 17 30180 19 31.5190 21 33.5200 22 34210 24 35220 25.5 36.5230 26 37240 27 38250 27 39260 27 38270 27 39280 27 39290 27 39300 27 39310 27 38320 28 37.5330 27.5 37340 27 37350 26 37.5360 26 37370 25 37380 24 36390 24 36400 24 34.5420 24 34440 23 33460 22.5 32480 22 32500 22 31.5520 21.5 31540 21 30560 20.5 31580 21 31600 21 30.5620 21 31640 21 30660 20.5 31680 21 31700 21 31720 21 31.5740 21 31760 21 31780 20.5 30.5800 20 30.5820 19.5 30840 19.5 30860 19 29.5880 18 29900 18 28920 18 28940 18 28______________________________________
The column is then eluted with methanol-0.05 N ammonium hydroxide. Fractions (20 ml) are collected and tested. Results follow.
______________________________________ Zone (mm)Fraction No. B. subtilis S. pyogenes______________________________________10 19 2920 18 28.530 18 2840 18 2850 18 2760 17 2670 16 2680 16 2690 16 26100 16.5 26110 16 25.5120 22 31130 28 38140 30.5 41150 28 38160 17.5 28.5170 0 20.5180 0 20.5190 0 20.5200 0 20220 0 18240 0 17.5260 0 17280 0 17300 0 18320 0 16.5340 0 16360 0 16.5380 0 17400 0 17.5420 0 16440 0 0. . .500 0 0______________________________________
The following combinations are made:
Pool IA: Fractions 170-400 (methanol-water 70:30)
Pool IIA: Fractions 401-950 (methanol-water 70:30) and 10-100 (methanol-0.05 N NH.sub.4 OH)
Pool IIIA: Fractions 115-170 (methanol-0.05 N NH.sub.4 OH)
Pool IA is concentrated to dryness. The residue obtained is triturated with 50 ml of methanol. The colorless insoluble crystalline acanthomycin is isolated by filtration and dried (ADA-49.1, 210 mg). The filtrate is mixed with ether-Skellysolve B (isomeric hexanes) (1:1) to yield additional amorphous acanthomycin (ADA-49.2, 500 mg).
Pool IIA, similarly, yields crystalline acanthomycin, prep. ADA-49.3, 220 mg and amorphous acanthomycin, prep. ADA-49.4, 300 mg.
Pool IIIA is concentrated to an aqueous solution and then freeze-dried to give acanthomycin ammonium salt (prep. ADA-49.5, 800 mg).

Antibiotic acanthomycin and process for preparing

Information

Patent Number

Date Filed

Date Issued

Inventors

Original Assignees

Examiners

Agents

CPC

US Classifications

Field of Search

US

International Classifications

Abstract

Description

Claims

US Referenced Citations (1)