Usage And Synthesis
The microorganism used for the production of antibiotic Actaplanin (A-4696)
has been identified as a strain of a species of Actinoplanes of the family
Actinoplanaceae. The Actinoplanaceae are a new family of microorganisms of
the order Actinomycetales, having been first described by Dr. John N. Couch,
Jour. Elisha Mitchell Sci. Soc., 65, 315-318 (1949).
The Actinoplanes sp. useful for the production of A-4696 was isolated from a sample of soil obtained from the Cascade mountain area in the state of Washington. Mycelial fragments of Actinoplanes sp., strain ATCC 23342 were inoculated on a nutrient agar slant having the following composition (g): pre-cooked oatmeal 60.0; yeast 2.5; K2HPO4 1.0; dried distiller's solubles 5.0; Czapek's mineral stock 5.0 ml; agar 25.0; water, deionized 1 L; Czapek's mineral stock has the following composition (g): KCl 100.0; MgSO4·7H2O 100.0; FeSO4·7H2O 2.0; (dissolve in 2 ml conc. HCl); deionized water 1 L. The slant was inoculated with ATCC 23342 and incubated for 6 days at 30°C. The culture does not normally sporulate on this medium, and it is necessary to macerate the mycelial mat with a flattened, sharpened, inoculating needle in order to increase the number of potential growth centers. The macerated mature culture was covered with sterile distilled water and scraped carefully with a sterile rod to obtain a mycelial suspension.
The suspension thus obtained was used to inoculate 100 ml of a sterile vegetative medium having the following composition (g): glucose 5.0; dextrin 20.0; soybean meal 5.0; yeast extract 2.5; calcium carbonate 1.0; tap water 1 L.
The inoculated vegetative medium was grown for 48 h at 30° on a rotary shaker operating at 250 rpm. 10 ml of the incubated vegetative medium was inoculated into 100 ml of a sterile "bump" medium of the same composition as given next above. The thus inoculated "bump" medium was incubated for 24 h at 30°C with constant shaking on a rotary shaker operating at 250 rpm. 0.4 ml of the incubated "bump" medium was inoculated into 100 ml portions of a production medium of the composition shown below contained in 500 ml. Erlenmeyer flasks, and sterilized at 120°C for 30 min (g): dextrose 1.0; dextrin 3.0; peptone 1.5; soybean meal 0.5; MgSO4·7H2O 0.2; molasses, beet sugar 1.5; corn steep liquor 0.5; betaine 0.1; K2HPO4 0.05; deionized water q.s. 25 L.
The pH of the medium was adjusted to 7.5 with 5 N sodium hydroxide solution before sterilization. After sterilization the pH was approximately 6.9. The production fermentation was shaken for about 96 h at a temperature of 30°C on a rotary shaker operating at 250 rpm. The pH at the end of the fermentation cycle was about 7.2.
The preparation of the inoculum proceeded through the incubation of the "bump" medium detailed above 25 L of a production medium as outlined above, with 0.02% Dow Corning antifoam added, was sterilized by autoclaving at 120°C for 30 min and charged into a 40 L fermentation tank. 100 ml of incubated "bump" medium was inoculated into the sterile production medium. The inoculated production medium contained in the 40 L tank was allowed to ferment for 4 days at 30°C. The fermentation was aerated with sterile air in an amount of about 0.5 volume of air per volume of culture medium per minute. The fermenting production medium was agitated with a mixer utilizing an impeller of a proper size and turning at an appropriate rpm to insure adequate mixing of air with the medium. The pH of the culture medium gradually increased from an initial level of about 6.9-7.2 as the fermentation proceeded.
The whole broth obtained from an A-4696 fermentation, was filtered with the aid of a commercial filter aid. The filtrate was set aside. The mycelial cake was washed with 32 L of water and the wash water set aside. The mycelial cake was then suspended in an additional 32 L of water and the pH of the mixture adjusted to pH 10.5 with 5 N sodium hydroxide solution. The mycelial cake water suspension was stirred for 45 min and the mixture was filtered. This filtrate and the water wash were combined with the original filtrate from the fermentation broth and the pH of combined filtrates was adjusted to pH 4.0 with H2SO4. The acidified combined filtrates was passed through a carbon column utilizing 1.0 kg of activated carbon, (Pittsburgh, 12x40). The activated carbon column was washed until the effluent was colorless. The A-4696 activity was adsorbed on the carbon column. The A-4696 activity was eluted from the carbon column utilizing a 1% H2SO4 solution in acetone: H2O (1:1). 2 L of the acidified acetone-water solution was sufficient to elute the A-4696 activity from the carbon column. The eluate containing the A-4696 activity as treated with a saturated barium hydroxide solution, in order to form a precipitate of barium sulfate, thus removing the sulfate ions from the solution. The mixture was filtered and the barium sulfate precipitate was discarded. The filtrate containing the A-4696 activity was concentrated under vacuum to dryness. The resulting residue comprising the A-4696 activity amounted to approximately 80.0 g.
The Actinoplanes sp. useful for the production of A-4696 was isolated from a sample of soil obtained from the Cascade mountain area in the state of Washington. Mycelial fragments of Actinoplanes sp., strain ATCC 23342 were inoculated on a nutrient agar slant having the following composition (g): pre-cooked oatmeal 60.0; yeast 2.5; K2HPO4 1.0; dried distiller's solubles 5.0; Czapek's mineral stock 5.0 ml; agar 25.0; water, deionized 1 L; Czapek's mineral stock has the following composition (g): KCl 100.0; MgSO4·7H2O 100.0; FeSO4·7H2O 2.0; (dissolve in 2 ml conc. HCl); deionized water 1 L. The slant was inoculated with ATCC 23342 and incubated for 6 days at 30°C. The culture does not normally sporulate on this medium, and it is necessary to macerate the mycelial mat with a flattened, sharpened, inoculating needle in order to increase the number of potential growth centers. The macerated mature culture was covered with sterile distilled water and scraped carefully with a sterile rod to obtain a mycelial suspension.
The suspension thus obtained was used to inoculate 100 ml of a sterile vegetative medium having the following composition (g): glucose 5.0; dextrin 20.0; soybean meal 5.0; yeast extract 2.5; calcium carbonate 1.0; tap water 1 L.
The inoculated vegetative medium was grown for 48 h at 30° on a rotary shaker operating at 250 rpm. 10 ml of the incubated vegetative medium was inoculated into 100 ml of a sterile "bump" medium of the same composition as given next above. The thus inoculated "bump" medium was incubated for 24 h at 30°C with constant shaking on a rotary shaker operating at 250 rpm. 0.4 ml of the incubated "bump" medium was inoculated into 100 ml portions of a production medium of the composition shown below contained in 500 ml. Erlenmeyer flasks, and sterilized at 120°C for 30 min (g): dextrose 1.0; dextrin 3.0; peptone 1.5; soybean meal 0.5; MgSO4·7H2O 0.2; molasses, beet sugar 1.5; corn steep liquor 0.5; betaine 0.1; K2HPO4 0.05; deionized water q.s. 25 L.
The pH of the medium was adjusted to 7.5 with 5 N sodium hydroxide solution before sterilization. After sterilization the pH was approximately 6.9. The production fermentation was shaken for about 96 h at a temperature of 30°C on a rotary shaker operating at 250 rpm. The pH at the end of the fermentation cycle was about 7.2.
The preparation of the inoculum proceeded through the incubation of the "bump" medium detailed above 25 L of a production medium as outlined above, with 0.02% Dow Corning antifoam added, was sterilized by autoclaving at 120°C for 30 min and charged into a 40 L fermentation tank. 100 ml of incubated "bump" medium was inoculated into the sterile production medium. The inoculated production medium contained in the 40 L tank was allowed to ferment for 4 days at 30°C. The fermentation was aerated with sterile air in an amount of about 0.5 volume of air per volume of culture medium per minute. The fermenting production medium was agitated with a mixer utilizing an impeller of a proper size and turning at an appropriate rpm to insure adequate mixing of air with the medium. The pH of the culture medium gradually increased from an initial level of about 6.9-7.2 as the fermentation proceeded.
The whole broth obtained from an A-4696 fermentation, was filtered with the aid of a commercial filter aid. The filtrate was set aside. The mycelial cake was washed with 32 L of water and the wash water set aside. The mycelial cake was then suspended in an additional 32 L of water and the pH of the mixture adjusted to pH 10.5 with 5 N sodium hydroxide solution. The mycelial cake water suspension was stirred for 45 min and the mixture was filtered. This filtrate and the water wash were combined with the original filtrate from the fermentation broth and the pH of combined filtrates was adjusted to pH 4.0 with H2SO4. The acidified combined filtrates was passed through a carbon column utilizing 1.0 kg of activated carbon, (Pittsburgh, 12x40). The activated carbon column was washed until the effluent was colorless. The A-4696 activity was adsorbed on the carbon column. The A-4696 activity was eluted from the carbon column utilizing a 1% H2SO4 solution in acetone: H2O (1:1). 2 L of the acidified acetone-water solution was sufficient to elute the A-4696 activity from the carbon column. The eluate containing the A-4696 activity as treated with a saturated barium hydroxide solution, in order to form a precipitate of barium sulfate, thus removing the sulfate ions from the solution. The mixture was filtered and the barium sulfate precipitate was discarded. The filtrate containing the A-4696 activity was concentrated under vacuum to dryness. The resulting residue comprising the A-4696 activity amounted to approximately 80.0 g.
Preparation Products And Raw materials
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