BE552077A - - Google Patents

Description

       

   <Desc / Clms Page number 1>
 



  The red, crystallized, anti-biotically active metabolic products of certain species of streptomycetes are known as Actinomycin's.



    According to: Waksman's research, streptomyces antibioticus provides Actinomycin A; Todd and his collaborators managed to isolate solutions

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 culture of another species of streptomyces,
 EMI2.1
 an Actinomycin to which they have called the name Actinomycin B; Brockmann. and his collaborators found as a product of the metabolism of strejjtomyees chrysoiaallus another jictinomycin to which they first gave the no. Actinochrysin, and then the name Actiriomycin C (see, on this subject German patent no.



  912, Ola of October 10, 1949). Brockmann succeeded in isolating from other strains of streptomyces yet two other Actinomycins which have been designated under the names Actinomycin J and Actinomycin X and which are apparently very close to Actinomycins of type A and 1 ;.



  Although Actinomycins behave similarly to
 EMI2.2
 unitary substances vis-à-vis conventional separation methods, it appeared in the partition analysis that they all consist of different components. A whole series of individual components of the various Actinomycins are currently known, which are identified by numerical indices. This is how, for example,
 EMI2.3
 that Acti2omycin C consists essentially of the components C., 'C2J and C3, and Actinomycins 1 "- and L also contain, in addition to the main component, still other secondary components.

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  Despite the multitude of theoretically possible combinations, it has so far appeared that the composition of the different Actinomycins is very characteristic as regards their components. Actinomycins type A and B, as well as Actinomycins J and X are made up essentially of one main component and in addition by distinct and characteristic secondary components, while Actinomycin C contains two main components, C2 and C, , a secondary component, C1, and in addition still infinitely small amounts of other Actinomycins.



  The composition of the different Actinomycins depends essentially on the species of streptomyces which was used for their production. The many strains examined so far can be divided into three distinct groups, namely strains producing type A and J respectively, strains forming type B and X respectively and finally strains forming type 0 (see this subject H.



    Brockmann, Angewandte Chemie, volume 66, 1, 1954).



  On the other hand, the composition of the Actinomycins was not influenced in appreciable proportions by the nutrient solution, - In the tests carried out so far, here we had not succeeded in essentially displacing the normal components by modifying the solution nutritive, and even less to cause the formation of new components which were not normally already present in the mixture.

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 EMI4.1
 



  According to the work of 14. Pfennig, Archiv f-or Likrobiologie, tome 18, 327., 1953), it is possible to suppose that the number of different etinomycira refocused in nature is not unlimited and that only there are a few distinct types of determined structure.
 EMI4.2
 In accordance with the present invention, it has been found that in the production of Actinomycins by culturing fungi strains selected from the group of str6p '"' A.lYO 'Capable of forming Actinomycins, the forma can be influenced. tion of the different components by adding to the media
 EMI4.3
 cultured nutrients, individual amino acids having more than two carbon atoms, or compounds capable of being converted to amino acids of this type under the conditions of cultivation.

   It is highly probable that these friends
 EMI4.4
 no-acids are incorporated into the Actinomycin molecule which, according to current knowledge, consists of a chromophore and a peptide-like side chain, and that the composition of the peptide chain undergoes a change when individual amino acids are present in very clear excess in the nutrient solution.
 EMI4.5
 



  Actir.or.rcins-generating agents had previously been cultivated on nutrient media containing dos 81i-i ..:, - o-ac: ide.s.



  For example, we had added to the solution YL; .t: C'î L1: i des.

 <Desc / Clms Page number 5>

 substances containing protein, such as soybean flour and yeast extracts, and glycocolle had also been used as a source of nitrogen.



  However, all these substances added for the purpose of improving the growth of the streptomyces strain did not influence the composition of the different components.



  This unexpected fact has been found that, for example, by adding valine to the nutrient solution of an Actinomycin C-generating agent, the formation of the main component C3 in favor of the formation of the component C1 and in this way making a secondary component a main component. In addition, we have made the even more surprising observation that it is even possible to obtain by way of biosynthesis new Actinomycins, hitherto unknown, as we have been able to observe, for example by adding isoleucine as the nutrient solution of an actinomycin generating agent, J. In addition to Actinomycin I, which has hitherto been obtained as the main component, a whole series of new and typical actinomycins is formed in this case in a predominant quantity compared to Actinomycin I.



   ! The new process therefore offers the possibility of preparing at will particularly desirable components as main components, or also of producing new, hitherto unknown Actinomycins which may be superior to known Actinomycins.

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  As amino acids which are added in accordance with the invention to nutrient solutions, not only those which are normally found in the peptide side chains of Actinomycins, such as, for example, valine, isoleucine, threonine, must be taken into account. , proline, sarcosine and N-methylvaline, but also those which. Until now, have not been found as structural ele- ments of Actinomycins, such as for example leucine, serine, arginine, N-methylalanine, as well as all the other amino -acids which in the levorotatory form normally constitute structural elements of peptide chains.

   As a rule, it suffices to add a single amino acid, but it has been found that the addition of games or more amino acids allows particular effects to be obtained in the composition of the Actinomycins formed.



  It is above all important that these additions generate a one-sided excess of individual amino acids in the nutrient solution and that in this way all amino acids are not equally accessible to streptomyces as this is done in the addition of albumioid substances .



  Instead of the amino acids themselves, it is also possible to add to the nutrient solution compounds capable of converting into amino acids under cultivation conditions, such as for example the keto and oxy acids which correspond

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 amino acids, or derivatives of amino acids such as, for example, their esters, amides or salts, or the alcohols corresponding to amino acids. You can also use droligo-. peptides to influence the formation of Actinomycins so that it continues in a specific direction.



  The oligopeptide is probably incorporated as such in the peptide chain, without being dissociated beforehand.



  The extent of influence exerted by amino acids depends on the concentration of added substances. If the concentration of the substances is relatively very low, practically no influence is observed; On the other hand, this influence is already manifested very clearly at concentrations of 0.3%, and it becomes very marked when the concentration of amino acids in the nutrient solution rises to 0.5% and more.

   As a general rule, it is advantageous to add the individual amino acids in a concentration of 0.3 to 1%, but it may sometimes be useful to employ other concentrations depending on availability and cost. Amino acid to be used In general, by the addition of individual amino acids, mixtures of different Actinomycins are also formed, which can be separated from the nutrient solution. according to known methods, for example by extraction with butyl acetate or ethyl acetate,

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 the composition of the different mixtures -d Actinomycins can be determined using the annular partition chromatography on paper.

   Actinomycins can be separated using partition chromatography, for example according to the process described in the patent application filed by the applicant on July 15, 1954 under no. 672,932, and isolated as pure individual components.



   Example 1: A nutrient solution of the following composition:
 EMI8.1
 
<tb> water <SEP> from <SEP> pipe <SEP> 1 <SEP> liter
<tb>
<tb> glycerin <SEP> 20 <SEP> g
<tb>
<tb> <SEP> potassium <SEP> nitrate <SEP> 5 <SEP> g
<tb>
<tb> D, L-valine <SEP> 5.8 <SEP> g
<tb>
<tb> <SEP> potassium <SEP> phosphate <SEP> secondary <SEP> 1 <SEP> g
<tb>
<tb> <SEP> sodium <SEP> chloride <SEP> - <SEP> 2 <SEP> g
<tb>
<tb> <SEP> calcium <SEP> carbonate <SEP> 1 <SEP> g
<tb>
<tb> magnesium <SEP> carbonate <SEP> <SEP> 1 <SEP> OR <SEP> mg
<tb>
<tb> bivalent <SEP> iron <SEP> <SEP> <SEP> <SEP> 40 <SEP> mg
<tb>
 (pH = 6.8-7.2) is inoculated with the streptomyces strain BoP 476 and shaken for 5 days at 30 ° C. in an Erlenmeyer flask.



  The culture solution having a final pH value of 8.4 and a .ctinomycin content of 24 mg per liter of solution of

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 The culture is extracted three times with stirring with a small amount of ethyl acetate or butyl acetate and the light red extract is concentrated under reduced pressure.

   The red residue washed with a small amount of hot ligroin provides in the ring partition chromatography, performed with the two-phase system: 1 part by volume of butanol, 4 parts by volume of di-n-butyl ether, 5 parts by volume of an 8% aqueous solution of sodium m-crsotinate, three rings with Rc values of 0.62,1.0 and 1.63 which correspond respectively to Actinomycins C1, C2 and C3. Actinomycin C1 is presented as the main component, Actinomycin C3 as a secondary component.



  We arrive at. same result using a nutrient solution of the following composition
 EMI9.1
 
<tb> water <SEP> from <SEP> pipe <SEP> 1 <SEP> liter
<tb>
<tb>
<tb> glycerin <SEP> 20 <SEP> g
<tb>
<tb>
<tb> .. <SEP> <SEP> potassium <SEP> nitrate <SEP> 6.6 <SEP> g
<tb>
<tb>
<tb> D, L-valine <SEP> 3.85 <SEP> g
<tb>
<tb>
<tb> <SEP> potassium <SEP> phosphate <SEP> secondary <SEP> 1 <SEP> g
<tb>
<tb>
<tb> <SEP> sodium <SEP> <SEP> 2 <SEP> g chloride
<tb>
<tb>
<tb> <SEP> calcium <SEP> carbonate <SEP> 500 <SEP> mg
<tb>
<tb>
<tb> <SEP> magnesium <SEP> carbonate <SEP> 50 <SEP> mg
<tb>
<tb>
<tb> bivalent <SEP> iron <SEP> <SEP> <SEP> <SEP> 10 <SEP> mg
<tb>
 (pH = 6.8 - 7.2).

   

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 the yield under the same culture conditions is 23 mg of Actinomycin per liter of culture solution, the final pH value after 6 days of incubation being 8. 4.



  On the other hand, the strain of streptomyces BoP 476 used in nutrient solutions free of valine, for example of the composition:
 EMI10.1
 
<tb> water <SEP> from <SEP> pipe <SEP> 1 <SEP> liter
<tb>
<tb>
<tb> glycerin <SEP> 20 <SEP> g
<tb>
<tb>
<tb> <SEP> potassium <SEP> nitrate <SEP> 10 <SEP> g
<tb>
<tb>
<tb> <SEP> potassium <SEP> phosphate <SEP> secondary <SEP> 1 <SEP> g
<tb>
<tb>
<tb> <SEP> sodium <SEP> <SEP> 2 <SEP> g chloride
<tb>
<tb> <SEP> magnesium <SEP> <SEP> 50 <SEP> mg
<tb>
<tb>
<tb> bivalent <SEP> iron <SEP> <SEP> <SEP> <SEP> 10 <SEP> mg
<tb>
   (pH = 6.8 - 7.2) provides a. mixture of Actinomycins, in which Actinomycins C3 and C2 are listed as main components and Actinomycin C1 as secondary component.



   Example 2: A nutrient solution of the following composition:
 EMI10.2
 
<tb> water <SEP> from <SEP> pipe <SEP> 1 <SEP> liter
<tb>
<tb> glycerin <SEP> 20 <SEP> g
<tb>
<tb> <SEP> potassium <SEP> nitrate <SEP> 5 <SEP> g
<tb>
<tb> D, L-isoleucine <SEP> 6.5 <SEP> g
<tb>
 

 <Desc / Clms Page number 11>

 
 EMI11.1
 
<tb> <SEP> potassium <SEP> phosphate <SEP> secondary <SEP> 1 <SEP> g
<tb>
<tb> <SEP> sodium <SEP> <SEP> 2 <SEP> g chloride
<tb>
<tb>, <SEP> calcium <SEP> carbonate <SEP> 1 <SEP> g
<tb>
<tb> magnesium <SEP> carbonate <SEP> <SEP> 100 <SEP> mg
<tb>
<tb> bivalent <SEP> iron <SEP> <SEP> <SEP> <SEP> 10 <SEP> mg
<tb>
 (pH = 6.8 - 7.2)

   is inoculated with the strain of streptomyces BoP 477 .. The culture solution is shaken for 5 days at 30 ° C. in an Erlenmeyer flask and treated as described in Example 1. The yield is 57 mg of Actinomycin per liter of culture solution, the final pH value is 8.3. Diaprés examination by ring partition chromatography, performed with the system:

   1 part by volume of butanol, 4 parts by volume of di-n-butyl ether, 5 parts by volume. From an 8% aqueous solution of sodium m-cresotinate, the isolated Actinomycin mixture consisted of approximately equal parts of five components having Rc2 values of 0.62, 1.0, 1.63, 2.41 and 3.53, On the other hand, the streptomyces strain BoP 477, used on nutrient solutions free of isoleucine, composed for example of
 EMI11.2
 
<tb> water <SEP> from <SEP> pipe <SEP> 1 <SEP> liter
<tb>
<tb> glycerin <SEP> 20 <SEP> g
<tb>
<tb> @arine <SEP> of <SEP> beans <SEP> of <SEP> soybeans <SEP> 5 <SEP> g
<tb>
 

 <Desc / Clms Page number 12>

 
 EMI12.1
 
<tb> glycocolle <SEP> 2,

  5 <SEP> g
<tb>
<tb>
<tb>
<tb> <SEP> potassium <SEP> phosphate <SEP> secondary <SEP> 1 <SEP> g
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> <SEP> sodium <SEP> chloride <SEP> 1 <SEP> g
<tb>
<tb>
<tb> <SEP> magnesium <SEP> <SEP> 100 <SEP> mg <SEP> sulfate
<tb>
<tb>
<tb>
<tb> Bivalent <SEP> iron <SEP> <SEP> <SEP> <SEP> 100 <SEP> mg <SEP>. <SEP>
<tb>
<tb>
<tb>
<tb>
<tb> <SEP> calcium <SEP> carbonate <SEP> 10 <SEP> mg
<tb>
 (pH = 6.8 - 7.2) forms according to the ring partition chromatogram (system indicated above) a mixture of Actinomycins consisting of a main component having the Rc value of 0.62 and a secondary component having the value Rc2 of 1.



  The Rc2 values are a measure which indicates the position of the respective zone in the annular partition chromatogram on paper, relative to the zone of the pure component C2 They are calculated by dividing the length of the path traveled by the component considered by the length the path traveled by component C2.



   Example 3 A nutrient solution of the following composition:
 EMI12.2
 
<tb> water <SEP> from <SEP> pipe <SEP> 1 <SEP> liter
<tb>
<tb>
<tb>
<tb> glycerin <SEP> 20 <SEP> g
<tb>
<tb>
<tb>
<tb> <SEP> potassium <SEP> nitrate <SEP> 5 <SEP> g <SEP>
<tb>
<tb>
<tb>
<tb>
<tb> D <SEP> L-isoleucine <SEP> 6.5 <SEP> g
<tb>
 

 <Desc / Clms Page number 13>

 
 EMI13.1
 
<tb> <SEP> potassium <SEP> phosphate <SEP> secondary <SEP> 1 <SEP> g
<tb>
<tb> <SEP> sodium <SEP> <SEP> 2 <SEP> g chloride
<tb>
<tb>
<tb> <SEP> calcium <SEP> carbonate <SEP> 1 <SEP> g
<tb>
<tb>
<tb> magnesium <SEP> carbonate <SEP> <SEP> 100 <SEP> mg
<tb>
<tb>
<tb> bivalent <SEP> iron <SEP> <SEP> <SEP> <SEP> 10 <SEP> mg
<tb>
<tb>
<tb> bacon <SEP> oil <SEP> <SEP> 1 <SEP> g
<tb>
 (pH 6.8 -7.2)

   is inoculated with the streptomyces strain BoP 476 and stirred for 5 days at 28 C in a submerged fermentation tank, under intense aeration. The culture solution having a final pH value of 8.9 is separated from the mycelium by a single centrifugation and then extracted five times, shaking with small amounts of ethyl acetate. The separated mycelium is also extracted with ethyl acetate until the extract shows a weak yellow color. The extracts are combined and the solvent is removed under reduced pressure. The red residue is washed twice with a small amount of hot ligroin.

   The yield of Actinomycin is 200 mg per liter of culture solution. To further purify this substance, the mixture is 'dissolved' in benzine, then filtered and adsorbed on **. aluminum oxide column. The red dye is eluted with a mixture of benzene and ethyl acetate (volume ratio 1) and recrystallized from pure ethyl acetate.



  Following the ring partition chromatography, performed

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 EMI14.1
 with the two-phase system: 1 part by volume of butanol, 4 parts by volume of dim-butyl ether, 5 parts by volume of an 8) aqueous solution; sodium d m-cresotinate, the Actinomycin mixture thus obtained is constituted in approximately equal parts of three components having the L c2 values of 1, 6, 2.4 and 3.5.



  In contrast, the streptomyces strain BoP 476, employed under similar submerged culture conditions on the same nutrient medium, but in the absence of isoleucine, provides a mixture of Actinomycins which, in ring partition chromatography (system above), consists of two main components with Rc2 values of 1 and 1.6. and a secondary component with Rc2 value of 0.62.



  Using a nutrient solution of the following composition:
 EMI14.2
 
<tb> water <SEP> from <SEP> pipe <SEP> 1 <SEP> liter
<tb>
<tb> glycerin <SEP> 20 <SEP> g
<tb>
 
 EMI14.3
 potassium nitrate 7 g
 EMI14.4
 
<tb> D, L-isoleucine <SEP> 1.5 <SEP> g
<tb>
<tb> <SEP> potassium <SEP> phosphate <SEP> secondary <SEP> 1 <SEP> g
<tb>
 
 EMI14.5
 c'iloride de sodium 2 g SL11: L'c: te de magnesium # 1c mag carbonate de Lcz "ilSlU.L'1 1CO Rb sulfate ds bivalent iron 10 iag

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 calcium carbonate 500 mg (pH = 6.8 - 7.2) is obtained with the same strain of streptomyces, with a yield of 150 mg of Actinomycin per liter of culture solution, a mixture of Actionmycins which, in as regards the main components, is identical with the mixture of Actinomycins described above.

   But according to the ring partition chromatography, carried out with the partition system: 4 parts by volume of di-n-butyl ether; 1 part by volume of butanol, 5 parts by volume of an 8% aqueous solution of sodium m-crsotinate, he will confide. besides the main components having the Rc2 values of 1.6, 2.4 and 3.7 still two secondary components having the Rc2 values of o, 62 and 1.0.



   Example 4 A nutrient solution of the following composition:
 EMI15.1
 
<tb>, <SEP> water.'de. <SEP> pipe <SEP> 1 <SEP> liter
<tb>
<tb> glycerin <SEP> 20 <SEP> g
<tb>
<tb> <SEP> potassium <SEP> nitrate <SEP> 5 <SEP> g
<tb>
<tb> D, L-leucine <SEP> 6.5 <SEP> g <SEP>
<tb>
<tb> <SEP> potassium <SEP> phosphate <SEP> secondary <SEP> 1 <SEP> g
<tb>
<tb> <SEP> sodium <SEP> <SEP> 2 <SEP> g chloride
<tb>
<tb> <SEP> calcium <SEP> parbonate <SEP> 500 <SEP> mg
<tb>
 

 <Desc / Clms Page number 16>

 
 EMI16.1
 .oa3! magnesium konate 100 mg.

   bivalent iron s, lf.tse 10 mg
 EMI16.2
 (pilaf 6e8 -r 7.2) is inoculated with the streptomyces strain BoP 476, (brewed, for 6 days at 28 0 in a submerged fermentation test, under intense aeration, and then tacitly subjected to describes the example The yield is
 EMI16.3
 of 65 mg of Actinomycin per liter of culture solution.



  According to the ring partition chromatography, carried out with the two-phase system: part by volume of butanol, 4 parts by volume of di-n-butyl ether, 5 parts by volume of an 8% aqueous solution of m -sodium cresotinate, the mixture of Actinomycins thus obtained is made up of four main components having the values
 EMI16.4
 jR of 1.0, '1, &, 2, 4 and 3, 5, and of' four secondary components having the R c2 values of 0.62, 1.2, 1.9 and 2.8.



  In contrast, the streptomyces strain BoP 476 provides under similar submerged conditions, but in the absence of
 EMI16.5
 leucine, a mixture of Actinomycins which, according to the ring partition chromatogram (system shown above), consists of two main components having the P2 contentions of 1.0 and 1.6, and 'a secondary component c2
 EMI16.6
 glove the R C2 value of 0.62.

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   Example 5: A nutrient solution of the following composition:

   
 EMI17.1
 
<tb> water <SEP> from <SEP> pipe <SEP> 1 <SEP> liter
<tb>
<tb>
<tb>
<tb>
<tb> glycerin <SEP> 20 <SEP> g
<tb>
<tb>
<tb>
<tb>
<tb> <SEP> potassium <SEP> nitrate <SEP> 5 <SEP> g <SEP>
<tb>
<tb>
<tb>
<tb>
<tb> D, L-norleucine <SEP> 6.5 <SEP> g
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> phosphate <SEP> .de <SEP> potassium <SEP> secondary <SEP> 1 <SEP> g <SEP>
<tb>
<tb>
<tb>
<tb>
<tb> <SEP> sodium <SEP> <SEP> 1 chloride,

  5 <SEP> g
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> <SEP> calcium <SEP> carbonate <SEP> 250 <SEP> mg
<tb>
<tb>
<tb>
<tb>
<tb> <SEP> magnesium <SEP> <SEP> 50 <SEP> mg
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> <SEP> magnesium <SEP> carbonate <SEP> 50 <SEP> mg
<tb>
<tb>
<tb>
<tb>
<tb> bivalent <SEP> iron <SEP> <SEP> <SEP> <SEP> 10 <SEP> mg
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> <SEP> baker's yeast <SEP> <SEP> (incubated <SEP> during
<tb>
<tb>
<tb> 3 <SEP> hours <SEP> to <SEP> 37 <SEP> C <SEP> before <SEP> the <SEP> sterilization
<tb>
<tb>
<tb> <SEP> of <SEP> the <SEP> nutrient <SEP> solution) <SEP> 500 <SEP> mg
<tb>
   (pH = 6.8-7.2) is inoculated with the streptomyces strain BoP 476 ot shaken for 6 days at 30 ° C. in an Erlenmeyer flask.



  The fermentation solution having a final pH value of 7.0 is separated from the mycelium by a single centrifugation and the resulting clear culture solution, together with the mycelium, is extracted with ethyl acetate or with butyl acetate. The extracts are combined and the dolvant is removed under reduced pressure. The wheel residue is completely extracted with benzene and adsorbed on a

 <Desc / Clms Page number 18>

 aluminum oxide column.

   The eluate obtained with a mixture of benzene and ethyl acetate (1: 2) gives, after concentration, with a yield of 36 mg per liter of culture solution, a mixture of Actinomycins which, according to ring partition chromatography (system: 1 part by volume of butanol, 4 parts by volume of di-n-butyl ether, 5 parts by volume of an 8% aqueous solution of sodium m-cresotinate ), consists of three main components with Rc2 values of 0.62, 1.0 and 1.63. The three components correspond to the components of the mixture of Actinomycins C1, C2 and C3.



  Under analogous test conditions, but in the absence of norleucine, strain BoP 476 also provides these three components, but in this case the component having the Rc2 value of 0.62 only manifests as a low secondary component. The addition of norloucine results in a shift in the ratio of the three components in favor of the component having the Rc2 value of 0.62.



   Example 6: A nutrient solution of the following composition:
 EMI18.1
 
<tb> water <SEP> from <SEP> pipe <SEP> 1 <SEP> liter
<tb>
<tb> glycerin <SEP> 20 <SEP> g
<tb>
<tb> <SEP> potassium <SEP> nitrate <SEP> 5 <SEP> g
<tb>
 

 <Desc / Clms Page number 19>

 
 EMI19.1
 
<tb> 'sarcosine <SEP> 4.4 <SEP> g
<tb>
<tb> <SEP> potassium <SEP> phosphate <SEP> secondary <SEP> 1 <SEP> g
<tb>
<tb>
<tb> <SEP> sodium <SEP> <SEP> 1 chloride,

  5 <SEP> g
<tb>
<tb>
<tb> <SEP> calcium <SEP> carbonate <SEP> 250 <SEP> mg
<tb>
<tb>
<tb> <SEP> magnesium <SEP> carbonate <SEP> 50 <SEP> mg
<tb>
<tb>
<tb> <SEP> magnesium <SEP> <SEP> 50 <SEP> mg
<tb>
<tb>
<tb> bivalent <SEP> iron <SEP> <SEP> <SEP> <SEP> 10 <SEP> mg
<tb>
<tb>
<tb> <SEP> baker's yeast <SEP> <SEP> (incubated <SEP> during
<tb>
<tb> 3 <SEP> hours <SEP> to <SEP> 37 <SEP> C <SEP> before <SEP> the <SEP> sterilization
<tb>
<tb> tion <SEP> of <SEP> the <SEP> nutrient <SEP> solution) <SEP> 2 <SEP> g
<tb>
 (pH = 6.8 - 7.2) est. inoculated with the streptomyces strain BoP 509, shaken for 6 days at 30 ° C., and treated as described in Example 5.

   The final pH value of the culture solution is 8.8. The yield of Actinomycin is 100 mg per liter of culture solution. According to the ring partition chromatography, carried out with the system: 5 parts and volume of amyl acetate, 5 parts by volume of a 10% aqueous solution of sodium m-cresotinate, the mixture of Actinomycins obtained consists of two main components having the 3 Rc2 values of 0.46 and 0.66, and a secondary component having the Rc2 value of 0.40.



    On the other hand, the BoP 509 strain employed under analogous culture conditions, in the absence of sarcosine, provides only a main component having the Rc2 value of 0.66 and a secondary component having the Rc2 value of 1.0.

 <Desc / Clms Page number 20>

 
Example 7:

   A nutrient solution of the following composition:
 EMI20.1
 
<tb> water. ' <SEP> of <SEP> line <SEP> 1 <SEP> liter
<tb>
<tb>
<tb> glycerin <SEP> 20 <SEP> g
<tb>
<tb>
<tb> <SEP> potassium <SEP> nitrate <SEP> 5 <SEP> g
<tb>
<tb>
<tb> sarcosine <SEP> 4.4 <SEP> g
<tb>
<tb>
<tb> <SEP> potassium <SEP> phosphate <SEP> secondary <SEP> 1 <SEP> g <SEP>
<tb>
<tb>
<tb> <SEP> sodium <SEP> <SEP> 2 <SEP> g chloride
<tb>
<tb>
<tb> <SEP> calcium <SEP> carbonate <SEP> 500 <SEP> mg
<tb>
<tb>
<tb> magnesium <SEP> carbonate <SEP> <SEP> 100 <SEP> mg
<tb>
<tb>
<tb> bivalent <SEP> iron <SEP> <SEP> <SEP> <SEP> 10 <SEP> mg
<tb>
 (pH = 6.8-7.2) is inoculated with the streptomyces strain BoP 476, shaken for 7 days at 30 ° C. in an Erlenmeyer flask and treated as described in Example 1.

   According to the annular partition chromatography, performed with the system: 1 part by volume of butanol, 4 parts by volume of di-n-butyl ether, 5 parts by volume of an 8% aqueous solution of m-cresotinate sodium, - the mixture of Actinomycin @ obtained with a yield of 30 mg per liter of * culture solution, consists of equal parts of five main components having R-values of 0.6, 0.8, 1.0 , 1.3 and 1.6, and a secondary component having the value Rc? from 0.45

 <Desc / Clms Page number 21>

 On the other hand, the streptomyces strain BoP 476, employed in the absence of sarcosine, provides a mixture of Actinomycins, in which the components having the Rc2 values of 1.0 and 1.6 are manifested as main components, and the components having the Rc2 value of 0,

  6 as secondary components
Example 8: A nutrient solution of the following composition. :
 EMI21.1
 
<tb> water <SEP> from <SEP> pipe <SEP> 1 <SEP> liter
<tb>
<tb>
<tb> glycerin <SEP> 2o <SEP> g <SEP>
<tb>
<tb>
<tb> <SEP> potassium <SEP> nitrate <SEP> 4.5 <SEP> g
<tb>
<tb>
<tb> N, N-dimethyldiketopiperazine <SEP> 9.0 <SEP> g
<tb>
<tb>
<tb> <SEP> potassium <SEP> phosphate <SEP> secondary <SEP> 1.0 <SEP> g
<tb>
<tb>
<tb> <SEP> sodium <SEP> <SEP> 2 chloride,

  0 <SEP> g
<tb>
<tb>
<tb> <SEP> magnesium <SEP> carbonate <SEP> 50 <SEP> mg
<tb>
<tb>
<tb> bivalent <SEP> iron <SEP> <SEP> <SEP> <SEP> 10 <SEP> mg
<tb>
<tb>
<tb> <SEP> calcium <SEP> carbonate <SEP> 500 <SEP> mg
<tb>
<tb>
<tb> <SEP> baker's yeast <SEP> <SEP> (incubated <SEP> during
<tb>
<tb> 3 <SEP> hours <SEP> to <SEP> 37 <SEP> 0 <SEP> before <SEP> the <SEP> strilisa-
<tb>
<tb> @tion <SEP> of <SEP> the <SEP> nutrient <SEP> solution) <SEP> 500 <SEP> mg
<tb>
 (pH = 6.8 - 7.2) is inoculated with the streptomyces strain BoP 509, shaken for 6 days at 30 ° C. in an Erlenmeyer flask and treated as described in Example 5.

   The yield is 34 mg of Actinomycin per liter of culture solution, the

 <Desc / Clms Page number 22>

 final pH value of the culture solution is 7.2. according to the 'ring partition chromatography, carried out with the system: 5 parts by volume of amyl acetate and parts by volume of an aqueous solution! 10% sodium m-cresotinate, the mixture of Actinomycins obtained consists of two main components having Rc2 values of 0.46 and 0.66.



  On the other hand, the streptomyces strain BoP 509 used in the absence of N, N'-dimethyldiketopiperazine, forms a mixture of Actinomycins consisting of a main component having the c2 value of 0.66, and a component secondary having the Rc2 value of 1.



   Example 9: A nutrient solution of the following composition.
 EMI22.1
 
<tb> water <SEP> from <SEP> pipe <SEP> 1 <SEP> liter
<tb>
<tb>
<tb>
<tb>
<tb> glycerin <SEP> 20 <SEP> g
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> <SEP> potassium <SEP> nitrate <SEP> 5 <SEP> g
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> <SEP> leucine <SEP> ethyl ester <SEP> hydrochloride <SEP> <SEP> 7.9 <SEP> g
<tb>
<tb>
<tb>
<tb>
<tb> <SEP> potassium <SEP> phosphate <SEP> secondary <SEP> 1 <SEP> g
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> <SEP> sodium <SEP> <SEP> 1 chloride,

  2 <SEP> g
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> <SEP> sodium <SEP> sulfate <SEP> 250 <SEP> mg
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> <SEP> magnesium <SEP> carbonate <SEP> 150 <SEP> mg
<tb>
<tb>
<tb>
<tb>
<tb> bivalent <SEP> iron <SEP> <SEP> <SEP> <SEP> 10 <SEP> mg
<tb>
 

 <Desc / Clms Page number 23>

 calcium carbonate 1 g baker's yeast (incubated for 3 hours at 37 C before sterilization of the nutrient solution) 1 g is added, before sterilization, of 0.4 cc of normal sodium hydroxide solution and then sterilized for one hour and a half at 110 C. After sterilization, the pH value is 6.5.

   The nutrient solution is inoculated with the BoP strain 476 and shaken in total for 5 days at 30 ° C. in an Erlenmeyer flask. During the two pre-. The first few days the pH value drops to 5.3, but after 5 days of incubation it reaches the value of 7.0. The culture solution is separated by centrifugation from the mycelium and the two fractions are thoroughly exhausted by extraction with ethyl acetate. The yield is 90 mg of Actinomycin per liter of culture solution. The extracts are combined and the solvent is removed under reduced pressure.



  The red residue is extracted thoroughly with benzene, filtered and adsorbed on an aluminum oxide column. The eluate obtained with a solution of benzene and ethyl acetate (1: 2) contains a mixture of Actinomycins which, according to the ring partition chromatography, carried out with the partition system: 6 parts by volume di-n-butyl ether: 1 part by volume of butanol and 7 parts by volume of a 6% aqueous solution of sodium m-cresotinate, consists of four main components having Rc2 values of 1 , 0, 1.25, @, 6 and 2.0, and a secondary component having the Rc2 value of 0.7.

 <Desc / Clms Page number 24>

 



  On the other hand, the streptomyces strain BoP 476 used in the absence of. Leucine ethyl ester hydrochloride, without sodium sulphate, but in the presence of a somewhat larger amount of sodium chloride, provides a mixture of Actinomycins which, according to ring partition chromatography, carried out with the system : 6 parts by volume of di-n-butyl ether, 1 part by volume of butanol, 7 parts by volume of a 6% aqueous solution of sodium m-cresotinate, consists of two components. principal having Rc2 values of 1.0 and 1.6, and a secondary component having Rc value of 0.7.



   Example 10: A nutrient solution of the following composition:
 EMI24.1
 
<tb> water <SEP> from <SEP> pipe <SEP> 1 <SEP> liter
<tb>
<tb> glycerin <SEP> 20 <SEP> g
<tb>
<tb> <SEP> potassium <SEP> nitrate <SEP> 5 <SEP> g
<tb>
<tb> D, L-leucine <SEP> 6.5 <SEP> g
<tb>
<tb> <SEP> potassium <SEP> phosphate <SEP> secondary, <SEP> 1 <SEP> g <SEP>
<tb>
<tb> <SEP> sodium <SEP> chloride <SEP> 1.5 <SEP> g
<tb>
<tb> <SEP> magnesium <SEP> <SEP> 50 <SEP> mg
<tb>
<tb> <SEP> magnesium <SEP> carbonate <SEP> 50 <SEP> mg
<tb>
<tb> bivalent <SEP> iron <SEP> <SEP> <SEP> <SEP> 10 <SEP> mg
<tb>
 

 <Desc / Clms Page number 25>

 
 EMI25.1
 
<tb> <SEP> calcium <SEP> carbonate <SEP> 250 <SEP> mg
<tb>
<tb> <SEP> baker's yeast <SEP> <SEP> (incubated <SEP> during
<tb>
 
 EMI25.2
 z hours z,

   57 C before sterilization
 EMI25.3
 
<tb> tion <SEP> of <SEP> the <SEP> nutrient <SEP> solution) <SEP> 2 <SEP> g
<tb>
 
 EMI25.4
 (pn = 6.8 7.2) is inoculated with the streptomyces strain BoP 509, washed for 8 days at 300 and treated as described in example 1. The final pH value of the solution of culture is 8.8. The yield of Actinomycin is
 EMI25.5
 of 30 mg per liter of culture solution.

   Next 1 "exc.- men by ring partition chromatography with the system; 4 parts by volume of di-n-butyl ether, 1 part by volume of butanol and 5 parts by volume of an 8% aqueous solution of sodium m-cresotinate, m-
 EMI25.6
 The isolated Actinomycin mixture consists of three main components having the S C2 values of 0.62, 1.0 and 1.5, and a secondary component having the Rc2 value of 2.2.



  On the other hand, the BoP 5o9 strain used in the absence of leu-
 EMI25.7
 cir - # ,,. ± orm a mixture of Actinomycins which, in the same ring-partition chromatogram, consists of a main component having the Rc 2 value of 0.62, and a secondary component having the So C2 value of, 0. ilxesiple '1 1 A nutrient solution of the following composition:

     
 EMI25.8
 
<tb> water <SEP> from <SEP> pipe <SEP> 1 <SEP> litr
<tb>
 

 <Desc / Clms Page number 26>

 
 EMI26.1
 
<tb> glycerin <SEP> 20 <SEP> g
<tb>
<tb>
<tb>
<tb> <SEP> potassium <SEP> nitrate <SEP> 5 <SEP> g
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> <SEP> <SEP> D hydrochloride, L-lysine <SEP> 7,

  3 <SEP> g
<tb>
<tb>
<tb>
<tb>
<tb> <SEP> potassium <SEP> phosphate <SEP> secondary <SEP> 1 <SEP> g
<tb>
<tb>
<tb>
<tb>
<tb> <SEP> sodium <SEP> <SEP> 2 <SEP> g chloride
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> <SEP> magnesium <SEP> carbonate <SEP> 5U <SEP> mg
<tb>
<tb>
<tb>
<tb>
<tb> bivalent <SEP> iron <SEP> <SEP> <SEP> <SEP> 10 <SEP> mg
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> <SEP> magnesium <SEP> <SEP> 10 <SEP> mg <SEP> sulfate
<tb>
<tb>
<tb>
<tb>
<tb> <SEP> calcium <SEP> carbonate <SEP> 500 <SEP> mg
<tb>
<tb>
<tb>
<tb>
<tb> <SEP> baker's yeast <SEP> <SEP> (incubated <SEP> during
<tb>
<tb>
<tb>
<tb>
<tb> 3 <SEP> hours <SEP> to <SEP> 37 C <SEP> before <SEP> the <SEP> sterilisa-
<tb>
<tb>
<tb> <SEP> of <SEP> the <SEP> nutrient <SEP> solution)
<tb>
 is added, before sterilization,

   1 cc of normal sodium hydroxide solution, then sterilized for an hour and a half at 110 C and inoculated with the BoP strain 476 and shaken for 5 days at 30 C. The final pH value of the culture solution is 6, 6 to 6.8. The yield of Actinomycin is 40 mg per liter of culture solution. The mixture of pure actinomycins is isolated as described in Example 3.



  According to the ring partition chromatography, carried out with: the system: 4 parts by volume of di-n-butyl ether, 1 part by volume of butanol, 5 parts by volume of an 8% aqueous solution of m-cresotinate sodium, the mixture of Action Mycins consists of two main components with Rc2 values of 1.0 and 1.6, and three secondary components with Rc 2 values of 0.6, 1. , 3 and 2, C

 <Desc / Clms Page number 27>

 On the other hand, the BoP 476 strain, used in the absence of lysine, forms a mixture of Actinomycins consisting of two main components having respectively the Rc2 value of 1.0 and 1.6, and of a secondary component having the Rc2 value of 0,

  6 (composition determined by ring partition chromatography using the system indicated above)
Example 12: A nutrient solution of the following composition:
 EMI27.1
 
<tb> water <SEP> from <SEP> pipe <SEP> 1 <SEP> liter
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> glycerin <SEP> 20 <SEP> g
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> <SEP> potassium <SEP> nitrate <SEP> 4 <SEP> g
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> D, L-leucylglycine <SEP> 9,

  3 <SEP> g <SEP>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> <SEP> potassium <SEP> phosphate <SEP> secondary <SEP> 1 <SEP> g
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> <SEP> sodium <SEP> <SEP> 2 <SEP> g chloride
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> magnesium <SEP> carbonate <SEP> <SEP> 100 <SEP> mg
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> <SEP> calcium <SEP> carbonate <SEP> 500 <SEP> mg
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> bivalent <SEP> iron <SEP> <SEP> <SEP> <SEP> 10 <SEP> mg
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> yeast '<SEP> from <SEP> baker <SEP> (incubated <SEP> during <SEP>
<tb>
<tb>
<tb>
<tb> 3 <SEP> hours <SEP> to <SEP> 37 <SEP> C <SEP> before <SEP> the <SEP> sterilization
<tb>
<tb>
<tb> <SEP> of <SEP> the <SEP> nutrient <SEP> solution) <SEP> 200 <SEP> mg
<tb>
 (pH = 6.8-7.2);

     is inoculated with the strain of streptomyces BoP 376 and shaken for 7 days at 30 ° C. in an Erlenmeyer flask.



  The culture solution with a final pH value of

 <Desc / Clms Page number 28>

 7.5 - 7.8 and giving a yield of 70 mg of Actinomycin per liter of solution, is treated as described in Example 5. Following the ring partition chromatography, carried out with the system: 6 parts in volume of di-nbutyl ether, 1 part by volume of butanol, 7 parts by volume of a 6% solution of sodium m-cresotinate, the resulting mixture of Actinomybines consists of a component secondary having the Rc value of 0.7 and four main components having the Rc2 values of 1.0, 1.25, 1.6 and 2.0.



  On the other hand, the streptomyces strain BoP 476, used in the absence of eucylglycine, forms a mixture of Actinomycins composed of two main components with the Rc2 values of 1.0 and 1.6, and of a secondary component having the value Rc2 of 0.7 (composition determined by ring-split c2 chromatography using the above system).



   'Example 13: A nutrient solution of the following composition t
 EMI28.1
 
<tb> water <SEP> from <SEP> pipe <SEP> 1 <SEP> liter
<tb>
<tb> glycerin <SEP> 20 <SEP> g <SEP>
<tb>
<tb> <SEP> potassium <SEP> nitrate <SEP> 5 <SEP> g
<tb>
<tb> D, L-threonine <SEP> 6 <SEP> g
<tb>
<tb> <SEP> potassium <SEP> phosphate <SEP> secondary <SEP> 1 <SEP> g
<tb>
<tb> <SEP> sodium <SEP> <SEP> 2 <SEP> g chloride
<tb>
 

 <Desc / Clms Page number 29>

 
 EMI29.1
 
<tb> <SEP> magnesium <SEP> carbonate.

   <SEP> 5o <SEP> mg
<tb>
<tb> <SEP> magnesium sulfate <SEP> 10 <SEP> mg
<tb>
<tb> Sulfate <SEP> of <SEP> iron <SEP> bivalent <SEP> 10 <SEP> mg
<tb>
<tb> <SEP> calcium <SEP> carbonate <SEP> 500 <SEP> mg
<tb>
<tb> <SEP> baker's yeast <SEP> <SEP> (incubated <SEP> during
<tb> 3 <SEP> hours <SEP> to <SEP> 37 <SEP> 0 <SEP> before <SEP> the <SEP> sterilization
<tb> of <SEP> the <SEP> nutrient <SEP> solution) <SEP> 500 <SEP> mg
<tb>
 (pH = 6.8 - 7.2) is inoculated with the streptomyces strain BoP 476, shaken for 7 days at 30 0 in an Erlenmeyer flask and then treated as described in Example 5. The pH value final culture solution is 8.3, the yield of Actinomycin is 70 mg per liter of culture solution.

   Following examination by ring partition chromatography carried out with the system: 4 parts by volume of di-n-butyl ether, 1 part by volume of butanol, 5 parts by volume of an 8% aqueous solution of m- sodium cre- sotinate, the resulting mixture of Actinomycins consists of one main component having the Rc2 value of 1.6, and two secondary components having the Rc2 values of 1.0 and 2.35. The main component 1,6 corresponds to Actinomycin C3.



  On the other hand, the streptomyces strain BoP 476, used in the absence of threonine, forms a mixture of Actinomycins consisting of two main components having the Rc2 values of 1.0 and 1.6, and a secondary component having the

 <Desc / Clms Page number 30>

 Rc2 value of 0.6 (composition determined by ring partition chromatography using the above system).



   Example 14 nutritional solution of the following composition:
 EMI30.1
 
<tb> water <SEP> from <SEP> pipe <SEP> 1 <SEP> liter
<tb>
<tb> glycerin <SEP> 20 <SEP> g
<tb>
<tb> <SEP> potassium <SEP> nitrate <SEP> 5 <SEP> g
<tb>
<tb> d, L-norvaline <SEP> 8 <SEP> g
<tb>
<tb> <SEP> potassium <SEP> phosphate <SEP> secondary <SEP> 1 <SEP> g
<tb>
<tb> <SEP> sodium <SEP> <SEP> 2 <SEP> g chloride
<tb>
<tb> <SEP> calcium <SEP> carbonate <SEP> 15u <SEP> mg
<tb>
<tb> <SEP> magnesium <SEP> carbonate <SEP> 5U <SEP> mg
<tb>
<tb> <SEP> magnesium <SEP> <SEP> 5u <SEP> mg
<tb>
<tb> <SEP> sodium <SEP> sulfate <SEP> 25 <SEP> mg
<tb>
<tb> bivalent <SEP> iron <SEP> <SEP> <SEP> <SEP> 25 <SEP> mg
<tb>
 (pH = 6.8 - 7.2) is inoculated with the streptomyces strain BoP 476,

   shaken for 5 days at 30 ° C. and treated as described in Example 5. The yield of Actinomycin is 35 mg per liter of culture solution. According to the ring partition chromatography, carried out with the system: 4 parts by volume of di-n-butyl ether, 1 part by volume of butanol, 5 parts by volume of a 3-fold aqueous solution; of

 <Desc / Clms Page number 31>

   Sodium m-cresotinate, the mixture of Actinomycins obtained consists of six components having the Rc2 values of 0.6, 0.75, 0.8, 1.0, 1.2 and 1.6.



  On the other hand, the streptomyces strain BoP 476 used in norvalin forms a secondary component having the Rc2 value of 0.6 and two main components having the Rc2 values of 1.0 and 1.6.



  Example 15: A nutrient solution of the following composition:
 EMI31.1
 
<tb> water <SEP> from <SEP> pipe <SEP> 1 <SEP> liter
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> glycerin <SEP> 20 <SEP> g
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> <SEP> potassium <SEP> nitrate <SEP> 4 <SEP> g
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> D,

  L-metthionine <SEP> 12 <SEP> g
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> <SEP> potassium <SEP> phosphate <SEP> secondary <SEP> g
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> <SEP> sodium <SEP> chloride <SEP> 2 <SEP> g <SEP>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> <SEP> calcium <SEP> carbonate <SEP> 250 <SEP> mg
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> magnesium <SEP> carbonate <SEP> <SEP> 5o <SEP> mg
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> magnesium sulfate <SEP> 50 <SEP> mg
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> <SEP> iron <SEP> sulfate <SEP> bivalent <SEP> 25 <SEP> mg '
<tb>
 baker's yeast (incubated for 3 hours at 37 C before sterilization of the nutrient solution) 200 mg (pH = 6.8 - 7.2)

 <Desc / Clms Page number 32>

 is inoculated with the streptomyces strain BoP 476,

   followed for 5 days at 30 ° C. in an Erlenmeyer flask and then treated as described in Example 5. The final pH value of the culture solutions is 7.4. The yield of Actinomycin is 63 mg per liter of culture solution. According to the annular partition chromatography, carried out with the partition system: parts by volume of di-n-buytl ether, 1 part by volume of butanol, 5 parts by volume of an 8% aqueous solution of Sodium m-cresotinate, the Actinomycins mixture consists of two main components with Rc2 values of 1.0 and 1.6, and a secondary component with Rc2 value of 0.6.



  On the other hand, the streptomyces strain BoP 476, used in the absence of methionine, forms a mixture of Actinomycins consisting of two main components having the Rc2 values of 1.0 and 1.6, and a secondary component having the Rc2 value of 0.6 (composition determined by ring partition chromatography using the system indicated above).



   Example 16: A nutrient solution of the following composition
 EMI32.1
 
<tb> water <SEP> from <SEP> pipe <SEP> 1 <SEP> liter
<tb>
<tb> glycerin <SEP> 20 <SEP> g
<tb>
<tb> <SEP> potassium <SEP> nitrate <SEP> 5 <SEP> g
<tb>
 

 <Desc / Clms Page number 33>

 
 EMI33.1
 
<tb>. <SEP> formyl-D-leucine <SEP> 8 <SEP> g
<tb>
<tb> <SEP> potassium <SEP> phosphate <SEP> secondary <SEP> 1 <SEP> g
<tb>
<tb> <SEP> sodium <SEP> chloride <SEP> 2 <SEP> g <SEP>
<tb>
<tb> <SEP> magnesium <SEP> <SEP> 50 <SEP> mg
<tb>
<tb> <SEP> magnesium <SEP> carbonate <SEP> 50 <SEP> mg
<tb>
<tb> bivalent <SEP> iron <SEP> <SEP> <SEP> <SEP> 10 <SEP> mg
<tb>
<tb> <SEP> calcium <SEP> carbonate <SEP> 250 <SEP> mg
<tb>
<tb> <SEP> baker's yeast <SEP> <SEP> (incubated <SEP> during
<tb> 3 <SEP> hours <SEP> to <SEP> 37 C <SEP> before <SEP> the <SEP> sterilization <SEP>

  of <SEP> the <SEP> nutrient <SEP> solution) <SEP> 500 <SEP> mg
<tb>
 (pH = 6.8 - 7.2) is inoculated with the streptomyces strain BoP 476, shaken for 4 days at. 30 C and treated as described in Example 5. The final pH value of the culture solution is 7.4, the yield of Actinomycin is 65 mg per liter of culture solution.

   According to the annular partition chromatography, carried out with the partition system: 4 parts by volume of di-n-butyl ether, 1 part by volume of butanol, 5 parts by volume of an 8% aqueous solution of m-cresotinate sodium, the mixture of Actinomycins' obtained consists of three main components having the Rc2 values of 1, u, 1.6 and 2.35, and two secondary components having the values of 0.6 and 3.4. ' In contrast, the streptomyces strain BoP 476, used without formyl-D-leucine, provides a mixture of Actinomycins

 <Desc / Clms Page number 34>

        consisting of 'two main components having the Rc2 values of 1.0 and 1.6, and of a secondary component having the Rc2 value of 0,

  6 (composition determined by ring partition chromatography using the same two-phase system).



   Example 17: A nutrient solution of the following composition:
 EMI34.1
 
<tb> water <SEP> from <SEP> pipe <SEP> 1 <SEP> liter
<tb>
<tb> glycerin <SEP> 20 <SEP> g
<tb>
<tb> <SEP> potassium <SEP> nitrate <SEP> 5 <SEP> g
<tb>
 
 EMI34.2
 aoetyl-DL # isoleucine 8.2 g
 EMI34.3
 
<tb> <SEP> potassium <SEP> phosphate <SEP> secondary <SEP> 1 <SEP> g
<tb>
<tb> <SEP> sodium <SEP> <SEP> 1 chloride,

  2 <SEP> g
<tb>
<tb> <SEP> calcium <SEP> carbonate <SEP> 500 <SEP> mg
<tb>
<tb> <SEP> magnesium <SEP> carbonate <SEP> 50 <SEP> mg
<tb>
<tb> Bivalent <SEP> iron <SEP> <SEP> <SEP> <SEP> 20 <SEP> mg
<tb>
<tb> Sodium <SEP> <SEP> <SEP> <SEP> 20 <SEP> mg
<tb>
 
 EMI34.4
 baker's yeast (incubated for * "1
 EMI34.5
 
<tb> 3 <SEP> hours <SEP> to <SEP> 37 C <SEP> before <SEP> the <SEP> sterilization- <SEP> 500
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> <SEP> tipn <SEP> the <SEP> nutrient <SEP> solution) <SEP> 500 <SEP> mg
<tb>
 is, before sterilization, adjusted to pH 7 by adding 2-normal sodium hydroxide solution, and the nutrient solution is inoculated with the streptomyces strain BoP 476 and shaken for 4 days at 30 ° C. in an Erlenmeyer flask.

   The

 <Desc / Clms Page number 35>

 nutrient solution then shows a pH value of 7.2. According to the ring partition chromatography, carried out with the two-phase system: 4 parts by volume of di-n-butyl ether, 1 part by volume of butanol, 5 parts by volume of an 8% aqueous solution of m -sodium crsotinate, the mixture of Actinomycins obtained with a yield of Actin-my.cine of 80 mg per liter of culture solution is made up of three main components having Rc2 values of 1.6, 2.4 and 3.5, and two secondary components having the values of 0.6 and 1.0.



  On the other hand, the streptomyces strain BoP 476 used without
 EMI35.1
 lacLty1 D, Z-.iolaue.ne, forms a mixture of Actinomycins consisting of two main components with Rc values of 1.0 and 1.6, and a secondary component with Rc2 value of 0.6 (composition determined by annular partition chromatography using the same two-phase system).



    ... Example 18: A nutrient solution of the following composition
 EMI35.2
 
<tb> water <SEP> from <SEP> pipe <SEP> 1 <SEP> liter
<tb>
<tb> glycerin <SEP> 20 <SEP> g
<tb>
<tb> <SEP> potassium <SEP> nitrate <SEP> 4 <SEP> g
<tb>
<tb> D, L-threonine <SEP> 4 <SEP> g
<tb>
<tb> D,

  L-valine <SEP> 4 <SEP> g
<tb>
<tb> <SEP> potassium <SEP> phosphate <SEP> 1 <SEP> g
<tb>
 

 <Desc / Clms Page number 36>

 
 EMI36.1
 
<tb> -Sodium <SEP> <SEP> <SEP> 2 <SEP> g
<tb>
<tb>
<tb>
<tb> <SEP> magnesium <SEP> carbonate <SEP> 50 <SEP> mg
<tb>
<tb>
<tb>
<tb>
<tb> <SEP> magnesium <SEP> <SEP> 25 <SEP> mg
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> bivalent <SEP> iron <SEP> <SEP> <SEP> <SEP> 25 <SEP> mg
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> <SEP> calcium <SEP> carbonate <SEP> 25U <SEP> mg
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> <SEP> baker's yeast <SEP> <SEP> (incubated <SEP> during
<tb>
<tb>
<tb> 3 <SEP> hours <SEP> to <SEP> 37 <SEP> C <SEP> before <SEP> the <SEP> sterilized # <SEP> 250
<tb>
<tb>
<tb> <SEP> of <SEP> the <SEP> nutrient <SEP> solution) <SEP> 250 <SEP> mg
<tb>
 (pH = 6.8 - 7.2)

     is inoculated with the strain of streptomyces BoP 476, shaken for 6 days at 30 C in an Erlenmeyer flask and treated as described in Example 5. The final pH value of the nutrient solution is 8.8, the Actinomycin yield is 40 mg per liter of culture solution. According to the experiment by ring partition chromatography, carried out with the two-phase system: 4 parts by volume of di-n-butyl ether, 1 part by volume of butanol, 5 parts by volume of an aqueous solution. 8% sodium m-cresotinate, the mixture of Actinomycins isolated consists of three main components having the Rc2 values of 0.6, 1.0 and 1.6, as well as a secondary component having the Rc2 value of 2.25.



  On the other hand, the streptomyces strain BoP 476, used in D, L-threonine and D, L-valine, forms a mixture of Actinomycins consisting of two main components having

 <Desc / Clms Page number 37>

 the Rc2 values of 1.0 and 1.6, and a secondary component having the Rc2 value of 0.6 (composition determined by ring partition chromatography using the same two-phase system).



   Example 19: A nutrient solution of the following composition:
 EMI37.1
 
<tb> water <SEP> from <SEP> pipe <SEP> 1 <SEP> liter
<tb>
<tb>
<tb>
<tb>
<tb> glycerin <SEP> 20 <SEP> g
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> <SEP> potassium <SEP> nitrate <SEP> 5 <SEP> g <SEP>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> D, L-valine <SEP> '<SEP> 6.5 <SEP> g <SEP>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> <SEP> potassium <SEP> <SEP> secondary <SEP> 1 <SEP> g
<tb>
<tb>
<tb>
<tb>
<tb> <SEP> sodium <SEP> <SEP> 2 <SEP> g chloride
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> magnesium <SEP> carbonate <SEP> <SEP> 100 <SEP> mg
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> sulfate, <SEP> of <SEP> magnesium <SEP> 10 <SEP> mg
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> bivalent <SEP> iron <SEP> <SEP> <SEP> <SEP> 10 <SEP> mg
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> carbonate,

   <SEP> of <SEP> calcium; <SEP> 1 <SEP> g
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>, .. baker's <SEP> <SEP> yeast <SEP> (incubated <SEP> during
<tb>
<tb>
<tb>
<tb> 3. <SEP> hours <SEP> to <SEP> 37 <SEP> C <SEP> before <SEP> the <SEP> sterilization- <SEP> 1
<tb>
<tb>
<tb> <SEP> tion <SEP> the <SEP> nutritive solution) <SEP> g
<tb>
 (pH = 6.8-7.2) is inoculated with the streptomyces strain BoP 458a, shaken for 5 days in an Erlenmeyer flask and treated in the manner described in Example 5. final pH value of the culture solution is 6.3, the yield of Actinomy-

 <Desc / Clms Page number 38>

 cine is 10U mg per liter of culture solution.

   Following the ring partition chromatography, carried out with the partition system: 5 parts by volume of amyl acetate, 5 parts by volume of a 1U% aqueous solution of sodium m-cresotinate, the mixture of Actinomycins thus obtained consists of a main component having the Rc2 value of 1.2, and two secondary components having the Rc2 values of 0.66 and 2.0 ..



  On the other hand, the streptomyces strain BoP 458a used without D, L-valine, forms a mixture of Actinomycins consisting of two main components having the Rc2 values of 0.66 and 1.2, and a secondary component having the Rc value 0.2 (composition, ¯ determined by ring partition chromatography using the partition system indicated above). The mixture of Actinomycins C shared with the system 5 parts by volume of amyl acetate, 5 parts by volume of a 10% aqueous solution of sodium m-cresotinate, provides two main components having Rc2 values of 1 (Actinomycin C2), 1.55 (Actinomycin C3) and a secondary component with an Rc2 value of 0,

  66 (Actinomycin C1). example 20 @ A nutritional solution of the following composition
 EMI38.1
 
<tb> water <SEP> from <SEP> pipe <SEP> 1 <SEP> liter
<tb>
<tb> glycerin <SEP> 20 <SEP> g
<tb>
 

 <Desc / Clms Page number 39>

 
 EMI39.1
 
<tb> <SEP> potassium <SEP> nitrate <SEP> 5 <SEP> g
<tb>
<tb> D,

  L-thrednine <SEP> 7 <SEP> g
<tb>
<tb> <SEP> potassium phosphate <SEP> secondary <SEP> 1 <SEP> g
<tb>
<tb> <SEP> sodium <SEP> <SEP> 2 <SEP> g chloride
<tb>
<tb> magnesium <SEP> carbonate <SEP> <SEP> 100 <SEP> mg
<tb>
<tb> <SEP> sodium <SEP> sulfate <SEP> 25 <SEP> mg
<tb>
<tb> bivalent <SEP> iron <SEP> <SEP> <SEP> <SEP> 10 <SEP> mg
<tb>
<tb> <SEP> calcium <SEP> carbonate <SEP> 1 <SEP> g
<tb>
<tb> <SEP> baker's yeast <SEP> <SEP> (incubated <SEP> during
<tb> 3 <SEP> hours <SEP> to <SEP> 37 <SEP> C <SEP> before <SEP> the <SEP> sterilization <SEP> of <SEP> the <SEP> solution <SEP> nutrient) < SEP> 1 <SEP> g
<tb>
 (pH = 6.8-7.2) is inoculated with the streptomyces strain BoP 458a, shaken for 6 days in an Erlenmeyer flask and treated as described in Example 5.

   The final pH value of the culture solution is 7.0, the yield of Actinomycin is 25 mg per liter of culture solution. According to the annular partition chromatography, carried out with the partition system: 5 parts by volume of amyl acetate, 5 parts by volume of a 10% aqueous solution of sodium m-crsotina, the mixture of Actinomycins thus obtained consists of two main components having the Rc2 values of 0.66 and 1.2, and four secondary components having the Rc2 values of 0.4, 1.0, 1.7 and 2, 1.



  On the other hand, the streptomyces strain BoP 458a used in the absence of D, L-threonine, forms a mixture of Actinomy-

 <Desc / Clms Page number 40>

 cines consisting of two main components having Rc2 values of 0.66 and 1.2, and a secondary component having Rc2 value of 0.2 (composition determined by ring partition chromatography using the same partition system).



   Example 21: A nutrient solution of the following composition:
 EMI40.1
 
<tb> water <SEP> from <SEP> pipe <SEP> 1 <SEP> liter
<tb>
<tb>
<tb>
<tb>
<tb> glycerin <SEP> 20 <SEP> g
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> <SEP> potassium <SEP> nitrate <SEP> 5 <SEP> g
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> sarcosine <SEP> 4,

  4 <SEP> g
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> <SEP> potassium <SEP> phosphate <SEP> secondary <SEP> 1 <SEP> g
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> <SEP> sodium <SEP> <SEP> 2 <SEP> g chloride
<tb>
<tb>
<tb>
<tb>
<tb> <SEP> magnesium <SEP> carbonate <SEP> 160 <SEP> mg
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> bivalent <SEP> iron <SEP> <SEP> <SEP> <SEP> 10 <SEP> mg
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> <SEP> calcium <SEP> carbonate <SEP> 1 <SEP> g
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> <SEP> baker's yeast <SEP> <SEP> (incubated <SEP> during
<tb>
<tb>
<tb> ..- <SEP> 3 <SEP> hours <SEP> to <SEP> 37 C <SEP> before <SEP> the <SEP> sterilization
<tb>
<tb>
<tb> tion <SEP> of <SEP> the <SEP> nutrient <SEP> solution) <SEP> 1 <SEP> g
<tb>
 (pH = 6.8 - 7.2) is inoculated with the streptomyces strain BoP458a,

   shaken for 8 days in an Erlenmeyer flask and treated as described in Example 5. The final pH value of the culture solution is 8.4, the yield of Actinomycin ect 25 mg per liter of culture solution. next

 <Desc / Clms Page number 41>

 ring partition chromatography, performed with the two-phase system: 5 parts by volume of amyl acetate, 5 parts by volume of a 10% aqueous solution
 EMI41.1
 of sodium m-cresotinate ', the mixture of actinomycins obtained consists of three main components having the
 EMI41.2
 Ro 2 values of 0.55e, 1.0 and 1.2, and five secondary components with Rc2 values of 0.2, 0.66, 1.55, 1.9 and 2.2.



  On the other hand, the streptomyces strain BoP 458a, used in the absence of sarcosine, forms a mixture of Actinomycins consisting of two main components having the R @ values of 0.66 and 1.2, and a secondary component having the 2 Rc2 value of 0.2 (composition determined by ring partition chromatography using the same partition system).



   Example 22: A nutrient solution of the following composition:
 EMI41.3
 
<tb> water <SEP> from <SEP> pipe <SEP> 1 <SEP> liter
<tb>
<tb> glycerin <SEP> 20 <SEP> g
<tb>
<tb> <SEP> potassium <SEP> nitrate <SEP> 5 <SEP> g
<tb>
 
 EMI41.4
 D, L'-isoleucine. 6-i 5 g
 EMI41.5
 
<tb> <SEP> potassium <SEP> phosphate <SEP> secondary <SEP> 1 <SEP> g
<tb>
<tb> <SEP> sodium <SEP> <SEP> 2 <SEP> g chloride
<tb>
<tb>
<tb> magnesium <SEP> carbonate <SEP> <SEP> 100 <SEP> mg
<tb>
<tb>
<tb> bivalent <SEP> iron <SEP> <SEP> <SEP> <SEP> 10 <SEP> mg
<tb>
 

 <Desc / Clms Page number 42>

 
 EMI42.1
 
<tb> <SEP> calcium <SEP> carbonate <SEP> 1 <SEP> g
<tb>
<tb> yeast- (incubated <SEP> during <SEP> 3 <SEP> hours <SEP> at
<tb> 37 <SEP> C <SEP> before <SEP> the <SEP> sterilization <SEP> of <SEP> the
<tb> nutrient <SEP> solution)

   <SEP> 1 <SEP> g
<tb>
 (pH = 6.8 - 7.2) is inoculated with the strptomyces strain BoP 458a, shaken for 5 days in an Erlenmeyer flask and treated as described in example 5. The pH value final culture solution is 7.5. According to the annular partition chromatography, carried out with the partition system: 5 parts by volume of amyl acetate, 5 parts by volume of a 10% aqueous solution of sodium m-cresotinate, the mixture of Actinomycins ob - held with a yield of Actinomycin of 70 mg per liter of culture solution, consists of eight components with Rc2 values of 0.66, 1.0, 1.2, 1.55, 1.9, 2.7 , 3.5 and 4.2.



  On the other hand, the s'treptomyces strain BoP 458a, used in the absence of D, L-isoleucine, forms a mixture of Actinomycins consisting of two main components having Rc2 values of 0.66 and 1.2, and of a secondary component
2 daire having the Rc2 value of 0.2 (composition determined by ring partition chromatography using the same partition system).

 <Desc / Clms Page number 43>

 
Example 23:

   A nutrient solution of the following composition: '
 EMI43.1
 
<tb> water <SEP> from <SEP> pipe <SEP> 1 <SEP> liter
<tb>
<tb>
<tb>
<tb>
<tb> glycerin <SEP> 20 <SEP> g <SEP>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> <SEP> potassium <SEP> nitrate <SEP> 5 <SEP> g
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> D,

  L-leucine <SEP> 8 <SEP> g
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> <SEP> potassium <SEP> phosphate <SEP> secondary <SEP> 1 <SEP> g
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> <SEP> sodium <SEP> <SEP> 2 <SEP> g chloride
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> magnesium <SEP> carbonate <SEP> <SEP> 100 <SEP> mg
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> bivalent <SEP> iron <SEP> <SEP> <SEP> <SEP> 10 <SEP> mg
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> <SEP> calcium <SEP> carbonate <SEP> 500 <SEP> mg
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> yeast <SEP> (incubated <SEP> for <SEP> 3 <SEP> hours
<tb>
<tb>
<tb> 37 <SEP> C <SEP> before <SEP> the <SEP> sterilization <SEP> of <SEP> the
<tb>
<tb>
<tb> nutrient <SEP> solution)
<tb>
   (pH ′ = 6.8 - 7.2) is inoculated with the streptomyces strain BoP 458a,

   followed for 5 days in an Erlenmeyer flask and treated as described in Example 5. The final pH value of the culture solution is 6.8, the yield of Actinomycin is 22o mg per liter of culture solution. According to the ring partition chromatography, carried out with the partition system: 5 parts by volume of amyl acetate, 5 parts by volume of an aqueous solution. At 10% sodium m-crsotinate, the mixture of Actinomycins obtained consists of four main components with Rc2 values of 0.66, 1,2,1,7, and 2.1, and three components se-

 <Desc / Clms Page number 44>

 condaries with Rc2 values of 0.1, 2.4 and 2.7.



  On the other hand, the streptomyces strain BoP 458a, used in the absence of D, L-leucine, forms a mixture of octinomycins consisting of two main components having the Rc2 values of 0.66 and 1.2, and a secondary component having an Rc2 value of 0.2 (composition determined by ring partition chromatography using the same partition system).


    

Claims (1)

Claim: Process for the production of Actinomycins by culturing strains of fungi selected from the group of streptomycetes capable of forming Actinomycins, characterized in that the nutrient medium used in the culture is added to individual amino acids comprising more than two carbon atoms, or compounds capable of being converted into amino acids of this type under culture conditions.