Chapter 5 



Isolation and Identification of 

 Antibiotics of Actinoniycetes 



Principles of Isolation 



The isolation of antibiotics of actino- 

 mycetes requires the use of methods which 

 have found general application in the isola- 

 tion of natural products. 



As will be discussed in detail in Chapter 6, 

 antibiotics of actinomycetes have extremely 

 di\Trse chemical structures. We find among 

 them acids, bases, amphoteric compounds, 

 neutral compounds, polypeptides, amino- 

 sugar complexes, compounds with huge 

 lactone rings, nitro compounds, guanido 

 compounds, polyenic compounds, and ace- 

 tylenic compounds. One cannot help being 

 amazed by the synthesizing capacities of 

 this group of organisms. 



Most antibiotics are released by actino- 

 mycetes into the culture medium; a few of 

 them are formed in the mycelium. Clearly 

 then, no single procedure will isolate all these 

 compounds. 



When one is dealing with an unknown sub- 

 stance, it is always advisable to have an idea 

 of the stability of the substance at various 

 temperatures and at \'arious pH \-alues 

 before attempting to devise a method of 

 extraction. A reliable assay is also necessary 

 to follow the fate of the antibiotic during 

 those various attempts. 



Since antibiotics are rarely formed alone 

 by the producing organism, but are often 

 elaborated as mixtures of complexes of re- 

 lated substances or along with totally unre- 



lated antibiotics, assay methods should give 

 not only quantitative data but also informa- 

 tion of a cjualitative nature. Qualitative data 

 permit one to follow the fate of the various 

 components of the complexes as they are 

 resolved by the extraction procedures. 



Some crude antibiotics are formed of 

 components which have a synergistic action. 

 Without proper bioassays, the activity would 

 seem to disappear during purification. This is 

 true of the antibiotic complexes E 129 and 

 PA 114. 



Basically there are two types of methods 

 for separating antibiotics from accompany- 

 ing impurities: (1) those which take advan- 

 tage of differences in solubility between com- 

 pounds and (2) those which take advantage 

 of differences in the affinity of such com- 

 pounds for the surfaces of adsorbents or ion 

 exchangers. Both methods liave endless 

 variations and are often used together in the 

 isolation of a given substance. 



The reader will find in Part B of this f)ook 

 outlines of the extraction and purification 

 procedures used for each one of the anti- 

 biotics described. He will note that for many 

 antibiotics, various methods of extraction 

 have been used. The (luality of the results 

 obtained with a given method will often 

 depend on the medium used for growing the 

 producing organism. Certain nutrients will 

 interfere with a given method of extraction, 

 iust as metabolites formed on a given me- 



40 



