BE719978A - - Google Patents

Description

  

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 EMI1.1
 



  Processes for the production of 2'-a, airo-2'-dEoxy-Icanamyc, in increased yield. The present invention relates to methods for producing
 EMI1.2
 with increased yields the '-am.zio-2'-esoxy-.canacnyc.ra which is. an antibiotic. More especially, relates to methods of producing 2-amino'-2-deso; Ky-it <: anMycin by fermentation and to methods of collecting and purifying this compound.
 EMI1.3
 



  2'-Amino-2'-deoxy-ltsunanycine is effective in supporting the growth of tiraln-positive, Gram-nat3vvs and aloldo-resistant bacteria, such as Staphylococcus, 9seuàomùnax, S1l11onl: l1a;

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 EMI2.1
 Shiolla. and Mycobacterium do mGLi, G that for 1nh1'Oer 'la c.:ro1:.;sa.nce
 EMI2.2
 do pathogenic bacteria which to antibiotics and agents
 EMI2.3
 cllil1jiothrE \ can1q, known synthetic uos. 2'-alUino..2 '... dso.xy "kanamycin has a very low toxicity and in particular 4zN little (side effects on the auditory system but a useful therapeutic effect on infections caused in' hoa: ae aminals by Gl'am-positive, Gra necative and acid-resistant bacteria.



  2 amino-2'-dësoxy-kanaNycin corresponds to the formula
 EMI2.4
 overall:
 EMI2.5
 Cl8 "370 'and to the structure oxwu7.a:
 EMI2.6
 
 EMI2.7
 'tuàa of the industrial production of kanaMyoine A by formentation, however, indicates that the ordinary natural strains of Streptoutyces lsanawyc; et.cus, used for the production of kanaisycin A on an industrial scale pe>' 1lot% in% of ' habàtude the fOI'tllation a 1> ùpottunte quuut1t of ltal1aycice a déuix the culture medium, but much less uno amount of 2-amino-2-dsoxy-kanamycin.
 EMI2.8
 



  Other reviews have indicated that it is possible to improve
 EMI2.9
 eor much the production and accumulation of 2 '.- aLina -'- dsorkanaliycin in the culture medium and isolate this compound with

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 EMI3.1
 a better yield either by cultivating the anuhet, o'dinairâr ao Streptomyces kanainycotîcuo in the usual way, united in parésenco of an aminoelucose or of a glucoziçilque compound ue 1'ûld..noglucose combined with de: 3ostl'eptUlliina, either by culturing certain new mutants of Streptomyces kanamyceticus in the conventional manner in an ordinary culture medium such as
 EMI3.2
 These actinomycotes of known types are usually used for cultivation.



   According to a first aspect, the invention has a pure object
 EMI3.3
 process for the production of 2'-amino-Z'-deoxy -kanany cne in increased yield, according to which a strain of
 EMI3.4
 Streptomyces kanamyceticus in an aerobic medium in a usual culture medium which contains a carbon hyurate and a nitrogenous food in the presence of at least one additive chosen from aminoglucoses and their glucosidic compounds combined with deoxy-
 EMI3.5
 streptamine, until 2'-am.no-2j-deoxy-kdnarvycin accumulates in substantial amount in the culture medium, then 2'-arnino-2'-deoxy-kanacr.ycir is isolated: te of the culture medium.



   As examples of the aminoglucoses which can be used in the process forming the first object of the invention, it is suitable:
 EMI3.6
 to cite D-glucosamine, 3-amino-3-dsoa.ynd.ucosc, 6 ... amino-6-deso.xy-D-glucoso and 2,6-ûi-cesox.y-2, , -uiaâülnG-Uglucose. By glucosidic compounds ae ll & min0elUCose combined with désaxystreptan: 3ne, there are compounds in which the Eom1noalucoso is combined with the side)., - yst'cpte, .n.lne UUl1S a ratio of 1 mole to 1 mole, or else complexes ;; of these olucosidic compounds associated with W1C another Uo glucose molecule or Ì.l / 1in061ucoses.

   Suitable examples of OOS glucosedic compounds of 11 aIúinoelucose combined with d4soyyztr * l) taLiric- are noted paromamine (or l- (2-Da; lucosa.nyl) -a-üi: snstnesp ,. tamine), desoÀ'Ystreptaine -O-karlosEúf.1nio.e "the neamine (or, - {2d (-di-deoxy-, b-diaâuino; lucosyl) -r.-désor.rstrc: pt:;, inc, 1>,

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 EMI4.1
 biotic zak-1001 (composed of 6-amino-6-o.6so.xy..D-Glucosa, do deoxysti, opt'viàno and do glucose; and the antibiotiquo DK-1003 (composa 0 6-azn.no- 6-dcsox, yy-L7 Llucose and deoxystroptauiin) (The antibiotics NK-l001 and NK-ldO, 3 above are new compounds which are the subject of Japanese patent applications Nos. 40145/67 and 40146/67 ( corresponding to English patent applications n
 EMI4.2
 28147/68 and 28148/68, caime specified above).

   In this process constituting the first object of the invention
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 tion, the aforementioned additive chosen from ilawinoglucose and the glucosidic compounds of 0'aninoglucose combined with deoxystreptamine, can be added to the culture medium before the cultivation of the stl'eptowyces, but this additive can also be introduced into the medium during culture, in a classical way this end. The proportion of additive in the culture medium
 EMI4.4
 is not critical and may be OeO5 to 1.0 by weight, but is preferably 0.1 to 0.5% by weight, based on the weight of the
 EMI4.5
 culture centre.

   It has been found that the amount of 2'-amino-2a-ddsoxy-kanmuycine produced and accumulated in the culture medium reaches its maximum after flow from 3 to 7 days after the start of the culture.
 EMI4.6
 The common strains of Streptomyces kanamyceticus now used for the industrial production of
 EMI4.7
 kanaMycin A have the ability to produce more 2'-amino 2'-deoxy..ltunamycin by culturing in the usual medium and in the arwcnco of the aminollucoses and of their decided derivatives; .ystreptam: 1.nQèS more,! llais on the contrary) as has been discovered, some npu, Vt.H1.U; '' mutants of Xtrcptouyces kanatuycoticus deriving from a typical 0-4-1 natural strain of Streptomyces kan / llJ / ycet.1cus may prove to be more rapid. of 2-aMinù-2-desoxy-kanaiiiycin even by culture in the usual medium and in the absence of azainoz;

  lucoses ot te 0 dÛSI?;.: ystl'epttiliniques d ': minobl, aforementioned ucoses, Cos new mutants which, by nature, have the ability

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 EMI5.1
 to produce more 2e-.wino-2e-ddsoxy-.M cine are obtained
From the known natural strain 0-4-1 of Streptomyces kana-
 EMI5.2
 yeeticus under the influence of an agent causing the mutation, namely nitrogen mustard or ultraviolet radiation and by subsequent special sorting of the surviving mycelia or spores.



   These new mutants who naturally have the ability
 EMI5.3
 to produce more 2'-arnino-2'-deoxy-kananycine are four of which received laboratory indices of A-4-6, 3AG, 18-8 and 19-2 respectively. These new mutants have in common the property of using xylose to a low or no extent, whereas the starting strains, on the contrary, have a greater ability to use xylose.



   The variation or mutation of Streptomyces kanamyceticus is a phenomenon which is not unexpected because this property is frequent in actinomycetes. Therefore, when any known strain of Streptomyces kanamyceticus is treated with an agent known to induce mutations such as nitrogen mustard, one can expect to obtain, in addition to mutants A-4-6 ,, 3AG, 18-8 and 19-2 above, still other mutants which
 EMI5.4
 do not have the property of producing more 2'-anino-2'-deoxyCanawr cine.



  According to the second of its aspects, llinventon therefore relates to a process for the production of 2'-amino-2'-deoxy-kanc: u: .. ycin with an increased yield according to which a mutant of Streptomyces kanamyceticus is cultivated. which naturally has the ability to produce more
 EMI5.5
 2'-amino-2'-deoxy-kanamycin aerobically in a usual culture medium containing the usual carbon and nitrogen sources until accumulation of 2'-amino-2'-deoxy-kanatnycine in a substantial amount in the culture medium, after which the antibiotic is isolated from the culture medium.



   According to this second aspect, the invention also has for
 EMI5.6
 subject of a process for the production of 2'-udno-2'-deoxy-kanamycin

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 with an increased yield according to which the mutant A-4-6, 3AG, 18-8 or 19-2 of Streptomyces kanamyceticus is carried out in an aerobic medium in a usual culture medium containing carbohydrates and nitrogenous nutrients, until accumulation of 2'-amino-2'-deoxy-kanamycin in a substantial amount in the culture medium, then this antibiotic is isolated from the culture medium.



   In the two methods constituting the first and the second aspect of the invention, the cultivation can be carried out in the usual manner according to the conventional methods for actinomycetes. Culture media containing the nutrients conventional for actinomycetes allow the production of 2'-amino-2'-deoxy-kanamycin. Thus, the culture medium may contain sources of carbon, such as starch, sucrose, maltose, glucose, glycerol, etc., and sources of nitrogen, such as soy flour, vegetable proteins. soluble, corn maceration liquor, peptone, meat extract, nitrates and ammonium salts.

   The culture medium may also contain other suitable inorganic salts, such as NaCl, MgSO4 and, if necessary, a substance promoting the growth of Streptomyces kanamyceticus.



   For the production of 2'-amino-2'-deoxy-kanamycin, it is possible to carry out the culture on solid medium but, for the production of large quantities, it. it is preferable that the cultivation takes place in a liquid medium under aerobic conditions.



  For large-scale production of the antibiotic, it is far better to resort to submerged culture with deep aeration. Culture with shaking or culture with agitation and aeration in liquid medium can be used. The culture temperature is usually 25 to 35 ° C but a preferred temperature is about 28 ° C.

   It has been found that the amount of 2'-amino-2'-deoxy-kanamycin which accumulates in the medium of

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 culture reaches its maximum after 3 to 7 days after the start of cultivation.
 EMI7.1
 The 2'-amino-2'-deoxy-kananycine which has accumulated in the culture medium can be isolated therefrom by one or the other conventional method involving ion-exchanged resins, by extraction with using organic solvents and by precipitation with the aid of a usual agent for the purification of known water-soluble basic antibiotics.

   For industrial production do
 EMI7.2
 2'-arnino-2'-deoxy-kanamycin, it is preferable to isolate the desired compound by adsorption and elution of an ion exchange resin, which is preferably a cation exchange resin of the type of carboxylic acids or by extraction with an organic solvent which contains a suitable vehicle, such as p-toluensulfonic acid or lauric acid.

   For example, we can iso-
 EMI7.3
 ler 2'-am.no - 2'-deoxy-lcanari; ycin by filtering off solids, for example mycelium, from fermentation broth containing 2'-amino-2'-dcso.y-kan: u > iycin, by adsorbing the filtrate on Ainberlite ll: ,, C-50 resin (ion-exchanging resin from Rohm & Haas CO, United States of America), eluting the resin with aqueous ammonia, by collecting and concentrating the active fractions, subjecting the concentrated fraction to chromatographic separation on Dowex 1 x 2 (ion exchange resin
 EMI7.4
 from Dow Cheinical C, USA from ADl61'ique), puiscncollcctt:., nt and concentrating the active fractions.

   The concentrated fraction was again subjected to a chlorine separation 1 tb raphiquc on Aeibarlite CG-50, after which a gradient elution was carried out with water and aqueous ammonia. We can obtain a uni-
 EMI7.5
 that containing an active compound, namely 2'-awino-2'-dôsoxykro1D.I: vrcine. This fraction is lyophilized and the resulting powder is recrystallized in a water mixture: to obtain the 2J-alll1n0-2'-deoxy-kanaJJvcinc in crystals.
 EMI7.6
 



  We. can get the 2'-a.i: o-2-co "ana: ycir.e under 14

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 EMI8.1
 form of acicular colorless crystals which grow at 183-186 C, which have a basic reaction and which are soluble in water, aqueous methanol, aqueous ethanol, afiueusc acetone, but which are insoluble in absolute methanol, (Absolute il, ethyl acetate, butyl acetate, ethyl ether, chloroform and benzene, which give a positive reaction to ninhy-
 EMI8.2
 drin: and to the reagents of Elson-Morgan and of Idiolisch, maize a negative reaction to the reagents of Sakaguchi, Tollens, Fehling, Millon ot Bénédicte a 1% aqueous solution of the substance having a
 EMI8.3
 optical rotation L-a-7D 21 more 118.

   An N-acetylato obtained by acetylation of 2'-amino-2 '.- âésohy-kanaLrcine using a mixture' of methanol and acetic anhydride has the overall formula C1SH32NO10.5 (CkiCO) .HZO , melts at 290-300 C with decomposition and has an optical rotation [[alpha]] 21 of plus 118.
D
 EMI8.4
 The antibacterial effect of 2 '- araino-2'-deoxy-kanarcine is not destroyed by heat. After a solution of 2'-amino-2'-esoxy-kanauiycin in 6N hydrochloric acid has undergone degradation by heating at 100 for 40 minutes, the antibacterial power against Bacillus subtilis of the degraded solution is still reduced. 60 to 80% of the power of the initial solution.

   The degraded solution was applied as a spot to a 40 cm x 40 cm sheet of Toyo No. 50 filter paper and subjected to upward development using a solvent which contained butanol, acetic acid and. water (4: 2: 1). This chromatography on paper shows that we obtain two spots, one of which gives a positive reaction.
 EMI8.5
 tiN'c to ninl1ydl'lnc and that one of the spots also gave a positive reaction to a: .uaoniacal silver nitrate.



  The 2'-a, uiuo-2'-désoy '-? Caxxa..ucinc has an antibacterial power'. 'N elové and useful against los Gram-positive and Gramn0otivos bacteria and 1-iùo-i, esistmites, ùe same as against ot strains bacteria is resistant to l: ana: cin A and other known antibiotics and chemotherapy agents. The toxicity of

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 EMI9.1
 2'-amino -: '.- desa.cy-lcnna; ycin; is very low ptl '! s'i'.4e "ev. - lethal doso 50 is barely: 190 mg / kg in mice (by intravenous injection).
 EMI9.2
 



  Table I below indicates the antibacterial spectrum of 2'-amino-2'-dso.cy-lttuza "ycin.



  T A 3 ia "¯ Çl Z,
 EMI9.3
 
<tb>
<tb> <SEP> micro-organisms tested <SEP> Minimum <SEP> concentration
<tb> for <SEP> inhibition
<tb>
 
 EMI9.4
 ¯ bzz¯ compl8to in? G / ll1
 EMI9.5
 
<tb>
<tb> Aerobactor <SEP> aerogenes <SEP> IAM <SEP> 1102 <SEP> 0.15
<tb> Alcaligenes <SEP> faecalis <SEP> 0.031
<tb>
 
 EMI9.6
 ] 3acillus agri 0,0? R3
 EMI9.7
 
<tb>
<tb> Bacillus <SEP> subtilis <SEP> ATCC <SEP> 6633 <SEP> oeo3g
<tb> Bacillus <SEP> subtilis <SEP> PCI <SEP> 219 <SEP> 0.078
<tb> Diplococcus <SEP> pneumoniae <SEP> Type <SEP> 311D <SEP> 0.31
<tb> Escherichia <SEP> coli <SEP> IAM <SEP> 1253 <SEP> 1.25
<tb>
 
 EMI9.8
 Klebsiella ptle umoniae 607 0, i
 EMI9.9
 
<tb>
<tb> Lactobacillus <SEP> arabinosus <SEP> ATCC <SEP> 8014 <SEP> 1.56
<tb> Lactobacillus <SEP> fermenti <SEP> ATCC <SEP> 9338 <SEP> 0.15
<tb> Staphylococcus <SEP> albus <SEP> PCI <SEP> 1200A <SEP> 0,

  039
<tb> Staphylococcus <SEP> aureus <SEP> 209P <SEP> 0.31
<tb> Staphylococcus <SEP> aureus <SEP> Terashima <SEP> 0.015
<tb>
 
 EMI9.10
 Mycobacterium phlei no 56 0.62
 EMI9.11
 
<tb>
<tb> Mycobacterium <SEP> phlei <SEP> Sekiguchi <SEP> 0.31
<tb> Mycobacterium <SEP> 607 <SEP> 0.78
<tb> Proteus <SEP> vulgaris <SEP> 7.8
<tb>
 
 EMI9.12
 Pseudomonas aoruginosa I1 1007 0.1 Pseudomonas aeruginosa Takasalci 0.78 Pseudoinonas aeruginosa alasakuro 0.39
 EMI9.13
 
<tb>
<tb> Salmonella <SEP> paratyphi <SEP> A <SEP> 0.62
<tb> Salmonella <SEP> paratyphi <SEP> B <SEP> 0.62
<tb> Salmonella <SEP> paratyphi <SEP> C <SEP> 1.25
<tb> Sarcina <SEP> lutea <SEP> PCI <SEP> 1001 <SEP> 0.31
<tb>
 

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 EMI10.1
 T A B L Ë A 0 i (cont.

   
 EMI10.2
 Microorganisms tested Minimum concentration
 EMI10.3
 
<tb>
<tb> for <SEP> inhibition
<tb> complete <SEP> in <SEP> g / ml
<tb>
 
 EMI10.4
 Sacclw, ro..1yces cerevisiae IAH 4626 500 SiiijeJ, the flexneri EW 10 2a o, 039
 EMI10.5
 
<tb>
<tb> Sreptococcus <SEP> faecalis <SEP> ATCC <SEP> 8043 <SEP> 15e6
<tb> Staphylococcus <SEP> aureus <SEP> 209P
<tb>
 
 EMI10.6
 (resisting to streptotiiycino) 2.5 Stap: ylococcus aureus 209P oe3l (resistant to \. h'ti.ll'O: llJrcine) 0.31 Eschorichia coli K-12 CS-2 Oe3l Escherichia coli K-12 Ci -2
 EMI10.7
 
<tb>
<tb> (resistant <SEP> to <SEP> antibiotics) <SEP> 1.56
<tb>
 In the drawings:

   Fig, Important on the y-axis the absorbance A and on
 EMI10.8
 abscissa the wavelength in mils, represents the absorption spectrum in the ultraviolet of 2'-animo-2'-deoxy-lcanaluycine in solution in water at a concentration of 1 mg / ml of 'water; Fig. 2, bearing in ordinates the transmission in prosecution
 EMI10.9
 hundred and on the x-axis the number of lorides in em-1, represents the infrared absorption spectrum of 2e-amîtio-Ze-d6so>: y-kanwayoine in Nujol.
 EMI10.10
 



  Coinine can be deduced from Figs. 1 and 2, 2'-amino-2'deoxy-katlavqrcine does not absorb in the ultraviolet -from 220 to 340: nillimicrons, but has characteristic absorption bands at the following number of waves: 3600, 3000, 1650 '1600, 1570, 1140;
 EMI10.11
 1110e 1095e 1090, 1060, 1035, 960e 900, 895e 880, 81.5a 815, 790, 780, 765, and 725 / cm-1.



   To carry out the method constituting the first aspect of the invention, it is possible to use a culture of the original strain
 EMI10.12
 0 -.- 1 of Streptomyces kanamyceticus which has been deposited in the permanent collection of microorganisms of the 1. T. C. C. in Washington, D.C. under No. 21252. In addition, cultures of mutants A-4-6,

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3AG, 18-8 and 19-2 of Streptomyces kanamyceticus which one can use in the process constituting the second aspect of E :! invention, have also been filed with the A.T.C.C. under nos. 21260, 21259, 21261 and
21268 respectively.



     @ As indicated above, the mutants A-4-6, 3AG, 18-8 and 19-2 of Streptomyces kanamyceticus are distinguished by a weak if not zero ability to use xyloso, unlike the strain d 0-4-1 origin which uses a lot of xylose.



   The mutant A-4-6 is distinguished, moreover, by the fact that its growth is null on a Czapek agar-agar plate with sucrose not containing deoxystreptamine, whereas the original strain 0-4- 1 can grow there. The 3AG sa mutant is distinguished by the fact that it forms much larger colonies than strain 0-4-1 on a Czapek agar plate containing 3-amino glucose. The 18-8 and 19-2 mutants used according to the invention are distinguished by the fact that they use dulcitol to a low or no extent whereas the original 0-4-1 strain can use much more dulcitol,
These mutants are produced by applying the following technique of mutation and sorting.

   The methods applied for each of the mutants A-4-6, 3AG, 18-8 and 19-2 are described in more detail below.



   The A-4-6 mutant is obtained by treating the 0-4-1 strain of Streptomyces kanamyceticus as follows: the typical strain of Streptomyces kanamyceticus above is brought by inoculation on the surface of an agar slant plate -czapek agar with sucrose and the culture is incubated at 28 ° C. for a period of 7 days. By means of a platinum loop, a fragment of the culture is taken and introduced into a liquid medium containing II *; corn maceration liquor and 0.25% dried yeast at pH 7.0 in a 70 ml test tube.



  Culture is run with shaking at 28 ° C for 24 hours

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 in a reciprocating device performing 350 cycles per Minute over an amplitude of 2 cm. The mycelium is isolated and grown by centrifuging the culture broth for 10 minutes under an acceleration of 2000 g and discarding the supernatant liquid. The mycelium cake is washed twice with equal amounts of 0.9% physiological saline solution, then the wet mycelium is ground in suspension in 0.9% physiological saline solution to a volume of 5 ml, then 4 ml of a solution containing 0.25 molar Na2HPO4 are added thereto,

   after which 1 ml of a 500 mg / ml solution of nitrogen mustard is added to the mixture, after which the mixture is left to stand for one minute. Then, 1 ml of the mixture is taken and added to 9 ml of a neutralization solution containing 0.2% glycine and 0.17% NaHCO3, after which the mixture is allowed to stand at room temperature for about 1 hour.

   By centrifugation, the mycelium is isolated which is rinsed and then transplanted whole into 1 ml of corn maceration liquor in a 70 ml test tube. Culture was performed by shaking at 28 ° C for 24 hours as described above. The culture product is rinsed twice, diluted and introduced by inoculation onto a sucrose Czapek agar plate further containing 100 mg / ml of deoxystreptamine. Incubation was carried out at 28 ° C. for 10 days to obtain colonies.



   A certain amount of mycelium is taken from each colony which is used to inoculate 10 ml of corn maceration liquor into 70 ml test tubes. Cultivation was again carried out by shaking at 28 ° C. for 24 hours, known above. The resulting cultures were rinsed twice, then or diluted and used to inoculate sucrose Czapek agar plates and sucrose Czapek agar plates containing greater than 100 mg /. ml ae deoxystreptamine. Incubation at 28 ° C for 7 days is performed to obtain colonies.

   We isolate '

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 a strain which does not grow on the first plate but only on the second and is called hereafter the A-4-6 mutant of
Streptomyces kanamyceticus. This mutant which consumes deoxystroptamine has an abundance of about one colony against 100.



   The 3AG mutant is produced by treating the original Shower 0-4-1 as follows: a culture of the typical strain 0-4-1 of Streptomyces kanamyceticus is applied by inoculation to the surface of an agar plate. -czapek agar with slanted sucrose and incubated for 7 days at 28 ° C. Using a platinum loop, a louse is removed from the culture and this amount is transplanted into 10 ml of a liquid medium containing 1% corn maceration liquor and 0.25% dried yeast at pH 7.0 in a 70 ml test tube.

   Cultivation was carried out by shaking at 28 ° C. for 24 hours in a reciprocating stirrer providing 350 cycles per minute with an amplitude of 2 cm. The culture is washed twice with physiological saline solution, then treated with 50 mg / ml nitrogen mustard solution. By means of mycelium of the strain thus treated, Czapek's medium containing 3% of 3aminoglucose instead of 3% of sucrose is inoculated, and the culture is carried out by shaking at 28 ° C. for 24 hours as above.

   The growing mycelium which is used to inoculate maize maceration liquor medium to enhance growth was separated from the culture by centrifugation. Cultivation was carried out by shaking again at 28 ° C. for 24 hours as above. The culture is then washed and the resulting suspension is homogenized and then diluted and used to inoculate 3-aminoglucose Czapek agar plates which are then incubated at 28 C for 14 days. to get colonies. Small, medium and large colonies are thus obtained.



  The mycelium is only collected from large colonies. and it is used to inoculate corn maceration liquor.

 <Desc / Clms Page number 14>

 
 EMI14.1
 



  Cn cx:; uLe? has culture it again at 2S '-' C during i! ./. nwu. ':; : '- "I' '4I ..; tút ... tJ..v: 1, C0 .. ::. 1C 1;., Ü: 11.1"'; above. After washing, the vN is homogenized; p; w 7: icn, diluted and unused to inoculate plates Gi'a, ;;,; t, t2 '^ :. "L: I' of C '.' .v: jC: i. to 3-mlnoglucose which is subjected to a new incubation at 28 C for 14 days, to obtain a new ".5 colonids. One thus obtains a strain giving only tol.on3.es of large dimension which one collects and which one calls ciur0s the'Mutant 3, au of Streptouyces kanamyceticus.



  The mutants 18-8 and 19-2 are obtained, the strain o: i..e 0-4-1 is treated as follows: by means of a culture of the original strain -4-1 of Streptooyces kanafayceticus, one The surface of a slanted Czapok agar plate is inoculated with ion and incubated at 28 ° C for 7 days.



  Using a platinum loop, a little of the culture is removed and this amount is suspended in 1 ml of sterilized water.



  After trituration, the product was transplanted into a polish containing starch Czapek's agur-a3ar and incubation was carried out at 2 ° C for 10 days.
 EMI14.2
 



  After incubation, 'we put the cup under a lamp
 EMI14.3
 ultraviolet irradiation (Toshiba C> 'Ja90n 19 watt sterilization lamp) and the ultraviolet irradiation is performed for 10 minutes while keeping the cup 30 cm from the lamp. After this wutation-inducing treatment, 10 n: yceliun 10 1 & surface of the α-agar is scraped and suspended in 10 ml of sterilized water. The suspeii-ion is homogenized, then it is not applied to 1> x1li, -agar of Csapek with duleitol and with af .: lidon (that is to say of .. 'slür -s3 jylr of Czapck containing 0.1 l / of starch ot 3 / J of 4ulcitol), then incubation is carried out at 23 ° C. for 7 days.

   The uyceliuu is only collected from small colonies which have grown weakly on J,. 'Arc "lax" This Ktyceliun is then used to inoculate the inclined plates of CZQe acar-aur to the a: hid which is subjected to incubation at

 <Desc / Clms Page number 15>

 
 EMI15.1
 28QO thought 7 days. All the colonies grew convinently on the αJ3x'-a; o.r plates tilted from Csapck to 1 "a :: dclcn.



  By means of (Puna elf loop one suparc the: ;; c <J1ium uo each tilted plate and one Unused to inoculate a plate included with aÛar-a; ar containing these mineral salts and bare dut ci toi (1 of dulc1 tol, 01264,3 of xaniniu sulfate <> 1, 0 .. 5Ó5,.; Of (, lroGl1ophosphate I.lipotaDs1que, 0, Z: 3S ,; do ctihyurosophosphato Monopotasulque, 0.1; of sulfate ae magnàsiluz hepta : y'dratb, 0.0006 / 1 '/' "ue sulphate cuivriquo pent, ai1yai'até, 010007 '; 1 / of chloride (..ú W ...' 1; MÙ: W t6tl'al1.Ydl ' até, 0.00011 ,,, da ferric sulfate hcpt: .ù1Yurat6, 0, Oü015 of heptal.'draé zinc sulfate and 1.5 ", of a; ar-ars pH 7.0).



  One incubation is carried out at 28 ° C. for 14 days to obtain a preponderant number of strains growing in the normal way as well as two strains which hardly grow sensitive.



  These two strains, which m.urr.irnt grew on the a; axwa'ar of Czrpr: lt at 1 "alidon" but not on the aax-aar containing salts Minerals: and dulcitol, have Inability to produce 2'-aràino-2? d6soxy-kro1cinc with increased efficacy and these two strains are hereinafter referred to as St1'0 mutants 1.3-8 and 19-2;) tO [, these kanaryceticus respectively.



  The mutants: 3G, A-4-6 "18-8 and 19-2 of 8trûtoLVC0 kanamyceticus which one uses according to the invention have 1., n; following characteristics: The uiutants 3ÂGy A-4-6, 18-3 and 1 -2 are Morphologically similar wis to others or cause the fl / C01: Lu ,;
 EMI15.2
 air gives ratifications, But not of a comparable finesse
 EMI15.3
 and does not usually give spirals and \ J vxi.l.ca ut uu; <= .t that born spores are i'o: ':. s i.'. l'cxtl'v ;;, itJ du. '. (..' t..l.;: aerial and usually have an oval or cylindrical shape with a width u, 1 .: 1.0 to 115 microns in G4CJi ü.;., 1 ;. surface these spores being .7 ....: i: 'st.



  However, these various strains are distinguishable 1; among others with regard to the conditions of growth on the

 <Desc / Clms Page number 16>

 
 EMI16.1
 C1C culture media, los C: .11? T ::: r physiological and 1 'pÉiÉé <io to
 EMI16.2
 Use of your carbon sources. The conditions of criL5Si.1l'lCO, the effects l) i1Y;: zi'Jlo.Lque:.; and the a;> ìt <i; 1 <v ii the uilixation dos IOU: r .'- ': C :; of mutant ceu curbone ue d i; v: ej) '4U'.,: jl,:, u l '.: ú, L1.UMycoti'jus t = have: r0:; Uús due..n ::: tables II, III and IV below.



  From plun) 1s> sx: iza't :: e ncr: between the typical strain 0-1r-1 da Streptor; yccs k: .ma: V'c0ticus and mutants 1 \ "'/ 1'-6 ,: 31.0, 18-8 and 19-2 which are derived therefrom are reviewed in Table IV below with respect to growth conditions, physiological effects and usability of carbon sources.

 <Desc / Clms Page number 17>

 
 EMI17.1
 



  T A B L E 1-. U II
 EMI17.2
 Conditions of growth on different culture media of mutants of Strept # ::; rces kanar.vceticv.s (observation after 1. days of incrbatîon at a temperature of 28 C)
 EMI17.3
 Culture llile-1 Observations Mutant A- ± -6 Mutant 3AG 1, lutant 18-8 hîutznt 19-2 1: ùcr-agar of c # ape3:

   Growth Uniform growth Uniform growth Uniform growth C-oi-sace 1X.'1.5 .. Sucrose - yellow staining yellow staining yellow staining l rnc coJ.oa tio! 1. shallow deep deep j a-g = + dark Zyc01i1X1 aerial Nil 'Nil Ileant î ", -' a P5.jtùc> iit solûDl e Weak yellow Weak yellow Yellow Weak yellow Revers Yellow coloring Yellow coloring ----- -----
 EMI17.4
 
<tb> grayish <SEP> low <SEP> grayish
<tb>
 
 EMI17.5
 J. . Growth Very uniform growth Uniform growth Uniform growth, iaeaiocre ...., coloration .......... faith, r; c glyccrol mediocre colorless coloration sris coloration Gr15 1 O! '::: C ccioraion yellowish yellowish brown elir liaiit Col l'yccliun acricn Nil Nil Nil Co'ratio :: j?, is oi '.' e lively soluble ii C "...



  PiC'-i6nt soluble Ncant Nil Nil Coorrti'ibrun
 EMI17.6
 
<tb> yellowish <SEP> weak
<tb>
 
 EMI17.7
 Reverse ----- Serious coloring Coloratic.njauriG ColorL.Gnbrun
 EMI17.8
 
<tb> weak <SEP> grayish <SEP> weak; <SEP> yellowish
<tb>
 

 <Desc / Clms Page number 18>

 
 EMI18.1
 T J. B E 1. U II (continuation 1)
 EMI18.2
 COZ1.! 'O "" <' "e '' '' -'-- 01r''C: -. '' '' *" I> è l: -r "d'1f") .. (. - :; t <- '-: - 11i ± "\' '' ''., .C: C1J1.t: .. ') C (2'" '"i1t ..."' t'- '" ('; (C; "".' T: J. '' '' ''. '"L ,.



  (o. ¯:> .... 1 .. "": -: - = 17 gir ....- ..J :. gwi s to 'µrr, 1 1,> 1 ï 1 -. J ¯. <; ..: ........: 1. a 1; 't Ul ...: ..-... \\ ....' 1 .... = nr "J <: f -,) 7 "" '"' f, -. U-'q: ":" 111 "." ':' 5 11. '01 -1-t "c. :: Ú '.L"' '' '' f \ 1 ". '' = - '- ; -: C1 "'" tl,} ""'; p i'C '""' \ .. 1 '. ("'. 'I" (' l. (, T- '"-: ..' O ("-" "'1o ...'.........-......-'., J- .10 .. -....,. V, - ,, -..., ---....., t¯v- "'" - ¯.¯-¯¯' -1 '. "J."' -.- -¯v¯- .... .. ¯ \ ... '-'.! .- - -. - - - ¯
 EMI18.3
 Medium of cciture Observations Mutant ù # - (- 6 l-uta! 1t 31G :: l.ltt:.: D ..; 1 .'... -.... r -.r ¯:, ai: F ::

   I., - o ¯Lk. :vs . .., J. [, 2¯! '- :: .. <:: r at Growth Colorc: .tion D:' 1ffi CO 03.'C: vlCxn J2'LL Coloration jl1 <lk '.UZ 6I'a . ', e C j;, i; iz <> i> aspii, a = 5¯ize and jlî:? vI''E3 a yellow jzi1 # iàtre deep deep vivid
 EMI18.4
 
<tb> to <SEP> glucose <SEP> deep <SEP> to <SEP> brown
<tb>
 
 EMI18.5
 (? = W¯; = -¯s: ") l.t ,. celi 121 aerial None i; W-.l: c Coloration j :: u == e l :(: r: t
 EMI18.6
 
<tb> weak
<tb> Pilent <SEP> soluble <SEP> none. <SEP> none <SEP> None <SEP> None
<tb> Reverse <SEP> Coloring <SEP> gray <SEP> Coloring <SEP> brown <SEP> ---- yellowish <SEP> weak
<tb>
 
 EMI18.7
 J - [; <: r - <: 1 [; & 1 'k Growth Staining j2.U: .r18 Staining jalJ21e Staining jclule Staining ja \:.!' .:

   1 '<:,; p2.ré: .Gine and deep to proforid yellow to pr-of yellow # 1d dark
 EMI18.8
 
<tb> to <SEP> glucose
<tb> (Uschinsky)
<tb> Mycelium <SEP> aerial <SEP> None <SEP> None <SEP> None <SEP> None
<tb> Pigment <SEP> soluble <SEP> None <SEP> None <SEP> None <SEP> None
<tb> Reverse <SEP> Yellow <SEP> coloring <SEP> Yellow <SEP> coloring '<SEP> ----- <SEP> ----
<tb> to <SEP> yellow <SEP> low <SEP> to <SEP> yellow <SEP> low
<tb>
 

 <Desc / Clms Page number 19>

   T A B L E A U II (continued 2) '
 EMI19.1
 Conditions what C O? S Sf..eCE 's'Ur Q1¯ ÎG'¯'G: l S Bilieux de c-LTitrre des suants de 5ti'epto = jccs}: C:,! 1:. ::, .- cct: .C ; .s CDSC'ilYWGIi after 1 day Cî? 12? Cl.iZJc:

  .'OI '? at a temperature of 28 C)
 EMI19.2
 Culture medium Observations Mutant fi -! - 6 Huant 3J.G 1.iutant lô-8 Mutant 19-2
 EMI19.3
 
<tb> Agar-agar <SEP> to <SEP> Growth <SEP> Color <SEP> yellow <SEP> Color <SEP> white <SEP> to <SEP> Color <SEP> white <SEP> to <SEP> Color <SEP> white <SEP> to
<tb> malate <SEP> of <SEP> calcium <SEP> weak <SEP> to <SEP> yellow <SEP> yellow <SEP> weak <SEP> .yellow <SEP> very <SEP> weak <SEP> yellow < SEP> very <SEP> low
<tb>
 
 EMI19.4
 î <h + c + aerial liIna. liena> it Nil Nil Nil Pit; :: Hmt sol1Jble Light coloring- Nil nil Nil
 EMI19.5
 
<tb> ment <SEP> yellow
<tb> Reverse <SEP> ----- <SEP> Coloration <SEP> white <SEP> Coloration <SEP> white <SEP> -----
<tb>
 
 EMI19.6
 ], c-r: .. r-t-C <1l '[.U Growth: rce Colorrtionbrun Co1or <:

  .tio! 1 brUl "i with Good growth Boncze c = * 1 <sin-e rllz.Lc <ic: calciun 'yellowish brown yellowish coloration yellow coloration yellow and with slycerol 1.iôrcélîim air None None None None Soluble pilent Yellow Yellow Yellow Ja1.U-; C îé: .ible
 EMI19.7
 
<tb> Reverse <SEP> Coloration <SEP> yellow <SEP> Coloration <SEP> brown <SEP> ---- weak
<tb>
 
 EMI19.8
 J, [; <:. 1 '- ::' (; <:. 1 'at the edge No colouration No colouration No colouration C ol a = + ati ccx no broth a crisatrous yellow a jà "m> e srisatre greyish yellow yellow gi, isi: t;> e
 EMI19.9
 
<tb> Mycelium <SEP> aerial <SEP> None <SEP> None <SEP> None <SEP> None
<tb> Pigment <SEP> soluble <SEP> None <SEP> None <SEP> None <SEP> None
<tb>
<tb>
 

 <Desc / Clms Page number 20>

 
 EMI20.1
 , 1 L D J, S L i3 ï 1 =.>? # Ûe 5 - - - - - - - - - -
 EMI20.2
 ,; ; ...-. "¯" = .¯; j. i =, =. dirf .- ..:.:;

   r¯g) j13.; =. = ± é = 1; ' 13'È'c:,: 1. "to" lzt¯ti from Strc - :. to-.yecG 1.lé: iiì.jcr "ï15-CU.S ('.' .: t '.:.: <: ':. i; j>, "Î" 1 /; jc "...;> r G' ± é # c1-, iti <; # i .... i: .¯ :. '': r: '' ..¯ ... 'ae 2o'C. / cr: s;': <, l:;:.: j 'ce: .c'c'.S
 EMI20.3
 
 EMI20.4
 r. -... 'w .- t. : '"i> -. (.. :: (¯:' '' :; ¯fC ': 1 .. as much I; -, ce-6 rlut? .. t'-t3LG." tl4e:? "t1 G v; ": 1.xt.c:. '. R! .T 19-2 .." -.... -.....' * -. - - ------- .. .. ¯ .. ¯ ¯. - ß G'¯: ... ¯.irG 'Cï'CJ'! .C.SF3: Ce l, '\ "r1 -"' '11;: .- Growth Zr. .¯'È'r fs: 'ç 31i Growth GLr E: - CTG? S3E .. =: CE G ..' rc-: '' '', -. ''. '... ¯¯ .. .¯. R id e colo: .ti # l ri .fe, colo! 'É.ti #' l Dcnt riùée, colo- u:! -. '.? ¯i. RQ COiO: <:. "'" 'Dlit.irt à e.là2'tE.' Weak bl: .nc to yellow low ration b2.anc to tien blanc a, l-, 4l:; rlf:

   
 EMI20.5
 
<tb> yellow <SEP> low <SEP> low
<tb>
 
 EMI20.6
 :: aerial yccli l: E: e.I'v t6 <; = Jt None None PiL -.- rit soluble None None None None 1 .. '::' - ';:'. ;. <; 2- Growth Staining yellow to Staining yellow-tr-.e Staining yellow Staining jccne ¯ ", ^ C ::: ... CI! ¯¯-. And yellowish-brown weak yellowish brown to f3ible yellow 2 yellow 1, l '(: z.:l:v1 ljr c 61 ij, aerial None none None None
 EMI20.7
 
<tb> Pilent <SEP> soluble <SEP> None <SEP> None <SEP> None <SEP> None
<tb>
 
 EMI20.8
 Reverse ----- ----- Yellow coloring Yellow coloring
 EMI20.9
 
<tb> weak <SEP> weak
<tb>
 
 EMI20.10
 Po: 1 :: 8 of land Growth Good growth Good growth.

   Raised growth, Raised growth, high, coloring surface gra-.iiilcv ,,:, e, grainy surface surface ± r21nÙeTICJ
 EMI20.11
 
<tb> ocher <SEP> coloring <SEP> brown <SEP> coloring <SEP> brown <SEP> coloring <SEP> brown
<tb> yellowish <SEP> weak <SEP> yellowish <SEP> weak <SEP> yellowish <SEP> weak
<tb>
 
 EMI20.12
 I¯'CE 3.1i7! .I airy White coloring Lej / # c- Light coloring- Nothing smells white Ment "1" white ....: Do, \; Dc ... C .. - ... Cl ... "G Iéaat PiG:, e :: - lt = oi ## 71st None None None None ...... 0 ¯. ¯- ¯¯¯¯ ¯¯.Jt # tt .- # Jt-.

 <Desc / Clms Page number 21>

 



    T A B L E A U II (continued 4)
 EMI21.1
 Growth cor.di tions SUl 'C: .. i CI'Gili: S nilieux de Ctùt1: .l'e mutants of StrCI} t # :. yCC: 5}: é:,; r.:ycE:tic1 : .s CLZri. '. = li ..'! G2? after 1, 'y days Ci? i ?? Ctitl'i0¯'1. at a tee: # Péiatlwe de 2BoC)
 EMI21.2
 Culture medium Observations ïulltêi2l 'A-.t-6 Hutant 3AG Ilutant lS-8. As much 19-2
 EMI21.3
 
<tb> Carrot <SEP> Growth <SEP> Wrinkled <SEP> Growth, <SEP> Wrinkled <SEP> Growth, <SEP> Wrinkled <SEP> Growth, <SEP> Poor <SEP> Growth
<tb> lesser <SEP> than <SEP> on <SEP> lesser <SEP> than <SEP> on <SEP> lesser <SEP> than <SEP> on
<tb> from <SEP> earth, <SEP> apple <SEP> from <SEP> earth, <SEP> apple <SEP> from <SEP> earth,

  
<tb> granular <SEP> surface <SEP> granular <SEP> surface <SEP> granular <SEP> surface
<tb> Mycelium <SEP> aerial <SEP> None <SEP> None <SEP> Staining <SEP> light- <SEP> None
<tb> ment <SEP> white
<tb> Pigment <SEP> soluble <SEP> None <SEP> None <SEP> none <SEP> None
<tb> Growth <SEP> to <SEP> 37 C <SEP> No <SEP> of <SEP> growth <SEP> No <SEP> of <SEP> growth <SEP> ----- <SEP> - ---
<tb>
 
 EMI21.4
 Srrü su sans de Growth - Growth of colo- Growth of colo- Growth of colo- C3'û: îûSF.'¯CE of colcLocffler gray to CrÉi.G 'ration gray to gray to CI'E :. ration r ..?. 't b ..

   C-3Ei: "t .trcéliu: aerial n none None None None l141 ;; = t Pi;: e: 2t soluble None None None None Growth a 37 C Similar to Similar to è # à> 1 òle àl.a - ---- growth at 28 C growth at 20 OC growth ±>. 2; JC 001.11 "Cr'OJ ': C: S'1" CC "O'Íc'c-"', "" c 2'W. É'.E. ', I.l'G: i .: ^ TtCcV ri; jE.E roissincl- C..éC :.

   E ..:., - !. ' yellow coloring yellow coloring co} o: ':' é. ';:;. 1.C1:;. ; Îi: ir.T '
 EMI21.5
 
<tb> Mycelium <SEP> aerial <SEP> None <SEP> None <SEP> None <SEP> None
<tb> Pigment <SEP> soluble <SEP> None <SEP> None <SEP> None <SEP> None
<tb>
 
 EMI21.6
 --------------------- -, - --- -------- ¯ "¯¯M-,. -.--- ¯. ---- '' '' ... r-

 <Desc / Clms Page number 22>

 
 EMI22.1
 '1.' 13 the;, j U -1-I (continuation 5)
 EMI22.2
 Coitl '; -:'. S ..; c craisi = #. 3 = .tù sr; '-' E.?¯ï 1 i''¯'E :: 7 è. $ NiH.or; -:; fl, # c;: 11:>: = * des i; iiii, tzi, s do Sl.;> r; j <; ¯;, c.¯i: CZ: C. '=': î.l. ''. "¯ '...., r):"' .... e- 1 /. days tl'ii # c; 1; z, 1 ± <, n to a * 1. ("" - '(,,.,:, .. 1..1' '' '(' '. of "j ......... t, '- # .--
 EMI22.3
 Medium d S C uù tià 'e OEJ S C 1 ia 1 to. O13 Hutant A - 4, - 6 iEl ité.iit 3 X.

   G II-Lmjit. 1 ± -S Jl 1 it. ; i = t ± j -2 LE.it ëcrcnë Growth No growth No growth Growth in é = rin "té, ii CY'V, i.l'i't, 1?, iàe in 8r; <1 / é; U 1 # JCé? .IC & aerial ----- ----- & nt i141: ià, PS-g% (. 13i SolïblÈ -----! En1t lï611ilt

 <Desc / Clms Page number 23>

   TABLE III Physiological effect of the mutant of Streptomyces kanamyceticus (observation after incubation for 14 days at 28 ° C.)

     
 EMI23.1
 
<tb> Physiological <SEP> Effects <SEP> Mutant <SEP> A-4-6 <SEP> Mutant <SEP> 3AG <SEP> Mutant <SEP> 18-8 <SEP> Mutant <SEP> 19-2
<tb> Reduction <SEP> of <SEP> nitrate <SEP> Positive <SEP> Positive <SEP> Positive <SEP> Positive
<tb> Hydrolysis <SEP> to <SEP> starch <SEP> Positive <SEP> Positive <SEP> Positive <SEP> Positive
<tb> Formation <SEP> of <SEP> sulphide <SEP> Negative <SEP> Negative <SEP> Negative <SEP> Negative
<tb> of hydrogen
<tb> Formation <SEP> of <SEP> tyrosinase <SEP> Negative <SEP> Negative <SEP> Negative <SEP> Negative
<tb> Liquefaction <SEP> of <SEP> the <SEP> gelatin <SEP> Strongly <SEP> liquefied <SEP> Liquefied <SEP> Liquefied <SEP> Liquefied
<tb> (at <SEP> 15 C <SEP> loser <SEP> 14 <SEP> days)

  
<tb> Solubilization <SEP> of <SEP> serum <SEP> to <SEP> No <SEP> observed <SEP> No <SEP> observed <SEP> No <SEP> observed <SEP> No <SEP> observed
<tb> blood <SEP> of <SEP> Loeffler <SEP> (at <SEP> 28 C <SEP> and
<tb> 37 C <SEP> for <SEP> 14 <SEP> days)
<tb> Medium <SEP> to <SEP> skimmed <SEP> milk <SEP> No <SEP> of <SEP> coagulation, <SEP> No <SEP> of <SEP> coagulation, <SEP> No <SEP> of <SEP> coagulation, <SEP> No <SEP> of <SEP> coagulation
<tb> peptonization <SEP> peptonization <SEP> slow peptonization <SEP> <SEP> slow <SEP> peptonization,
<tb> fast <SEP> fast
<tb>
 

 <Desc / Clms Page number 24>

 
 EMI24.1
 2 1-. J3 -.- k.JG \ JI-LY.
 EMI24.2
 



  D-i. "'! ..': lf" :: "S.:? ::: - = :: 1 to";!: Tjr; i ::,. 1.l- :? 0-1 ..- 1 and the 1: 11t! 1t.S dB St: trJtf) w, rces 1 '>; - x- = r. "I; iaus
 EMI24.3
 i's: 3r'é: .. ". = 's, ± y :: 5 Strain O- ± -1 t7 3: u! i; # I'.L r¯-.ir-r3' 'Mutant 3AG iéutz¯ nt 18-3 as much 19-2 6'.ZGJ¯C¯; ¯¯ Plate .a '4fGi ¯x-4G3 of CzàPeÉ T 1 i' - -H- + 4t, A S..CCil.06 Plate = ' * ra = - * ca = - C: e 'Cza? ek' '-iw la dcso.:-yEtr&pt:ii:e Czapek agar-e & r plate - -F --- I- at 3's.i. : '1.r0 utL .'.- e Czapek agar-agar plate ... iii- ++' wi- - ¯i
 EMI24.4
 
<tb> to <SEP> dulcitol
<tb>
 
 EMI24.5
 P13T e de 'terre Wrinkled growth Good growth Growth r3.C.E growth, Wrinkled growth, area ßa2tei:

  .5e, high, colored, sur- surface granule'c. - surface granucoloration slightSteiit tion ocher face granule, colouration Icuse, colorabrune, colo- yellowish brown tien. yellow brown weak brown natural weak
 EMI24.6
 
<tb> yellowish
<tb> ------ <SEP> low <SEP>
<tb>
 
 EMI24.7
 Effect; sio3 oic; e
 EMI24.8
 
<tb> Liquefaction <SEP> of <SEP> the <SEP> gelatin <SEP> +
<tb>
 
 EMI24.9
 Hilieu with skimmed milk Slow peptonization Peptonization Peptonization Peptonization Pepé "or-isa4-i-on r4p ce fast slow slow

 <Desc / Clms Page number 25>

   T A B L E A U IV (continued)
 EMI25.1
 ¯¯¯¯¯¯¯Differentiate these ez% g = e the strain t-.:!pīO-4-1 and the mutants of Stre..pt: l1Ls ks1:?:. ::

  rr¯eticus
 EMI25.2
 
<tb> Observations <SEP> Strain <SEP> 0-4-1 <SEP> typical <SEP> Mutant <SEP> A-4-6 <SEP> Mutant <SEP> 3AG <SEP> Mutant <SEP> 18-8 <SEP> Mutant <SEP> 19-2
<tb> <SEP> use of source <SEP>
<tb> of <SEP> carbon
<tb> Sucrose <SEP> +++ <SEP> # <SEP> ++ <SEP> + <SEP> +
<tb> Xylose <SEP> ++ <SEP> - <SEP> - <SEP> # <SEP> #
<tb> <SEP> sodium acetate <SEP> ++ <SEP> - <SEP> # <SEP> +++ <SEP> +
<tb> Citrate <SEP> of <SEP> sodium <SEP> +++ <SEP> + <SEP> ++ <SEP> + <SEP> +
<tb> Suçcinate <SEP> de <SEP> sodium <SEP> + <SEP> # <SEP> ++ <SEP> + <SEP> +
<tb>
 
 EMI25.3
 Dulcitol +++ ++ +++ # #

 <Desc / Clms Page number 26>

 
Antibiotics NK-1001 and NK-1003 which can be used as an additive in the process constituting the first aspect,

     of the invention described above are novel antibiotics which can be obtained by culturing a strain of Streptomyces kanamycicus in a medium containing a special additive.



   Antibiotic NK-1001 is a new substance which is effective in inhibiting the growth of Gram-positive and Gram-negative bacteria like Bacillus subtilis, Lacto-bacillus fermenti and Shigella dysenteriae, which is basic and soluble in water, methanol aqueous and aqueous ethanol, but insoluble in absolute methanol, absolute ethanol, ethyl acetate, butyl acetate and benzene, which gives a positive reaction with ninhydrin and with Elson's reagents --Morgan and Molisch, but a negative reaction with the reagents of Sakaguchi, Tollens, Fehling, Millon and Benedict, which contains carbon hydrogen, oxygen and nitrogen, but does not contain sulfur, d 'halogens and ash,

   which corresponds to the global formula C18H35N3O12.H2O and which forms colorless needle crystals decomposing at 238-242 C, which in aqueous solution shows a rotatory power [[alpha]] D21 more than 132, which does not absorb in the ultraviolet from 220 to 340 millimicrons as shown in Fig. 3 showing the absorbance A on the ordinate, the wavelength on the abscissa and which has characteristic absorption bands in Nujol in the infrared region as shown in FIG.

   4 showing the transmission in percent on the ordinate and the number of waves on the abscissa. The NK-1001 antibiotic hydrolyzed by heating in solution in 6N hydrochloric acid for 40 minutes at 100 C loses its antibacterial activity. The resulting hydrolysis solution is applied as a spot on a ribbon (40 cm x 2 cm) of Toyo No. 50 filter paper, then developed in descending bran using a mixture of butanol-pyridino-acetic acid. -water (6:.: 1: 3).

   The resulting paper chromatogram shows the

 <Desc / Clms Page number 27>

 formation of three spots, namely one which gives positive reactions to ninhydrin and ammoniacal silver nitrate, the other which gives a positive reaction to ninhydrin only and the third which gives a positive reaction to ammoniacal nitrate. ammoniacal silver. From the comparison with standards, it can be deduced that these three spots correspond to deoxy streptamine, 6-amino-6-deoxy-D-glucose and D-glucose, respectively.

   For this reason and knowing the molecular weight of the antibiotic NK-1001, we can consider that the antibiotic NK-1001 is a new compound consisting of a molecule of deoxy streptamine, a molecule of 6-amino-6-deoxy -D-glucose and a D-glucoso molecule. The antibiotic NK-1001 is substantially non-toxic since its lethal dose when administered intravenously in mice is 3050 mg / kg.



   Antibiotic NK-1001 can be produced by culturing the above typical strain 0-4-1 of Streptomyces kanamyceticus under aerobic conditions in a culture medium containing a carbohydrate and nitrogenous food, as well as some amount of streptidin, until the antibiotic NK-1101 builds up in the medium, then isolating the antibiotic from the culture. The technique for producing the antibiotic NK-1001 by fermentation and the nature of the carbon and nitrogen sources may be similar to the techniques and sources suitable for the production of 2'-amino-2'-deoxy-kanamycin, such as described above. It is therefore possible to apply agitation culture and tank culture. The culture temperature can be 25-35 C and is preferably about 28 C.

   The amount of streptidine added to the culture medium is not critical but is economically 0.05 to 1.0% and preferably 0.1 to 0.5% by weight of the medium. Streptidin can be added at the start of cultivation or during the operation itself.

 <Desc / Clms Page number 28>

 



   Isolation and purification of the antibiotic NK-1001 can be performed known for 2'-amino-2'-deoxy-kanamycin, as described above.



   The antibiotic NK-1003 which can be used as an additive like the antibiotic NK-1001 is also a new antibacterial substance effective against Gram-positive, Gram-negative and acid-fast bacteria like Bacillus subtilis, Lactobacillus fermenti, Shigella dysenteriae and so on.



  Antibiotic NK-1003 is a substance which is effective in inhibiting the growth of Gram-positive, Gram-negative and acid-fast bacteria which is basic and soluble in water, aqueous methanol and aqueous ethanol but insoluble. in absolute methanol, absolute ethanol, ethyl acetate, butyl acetate and benzene, which gives a positive reaction to ninhy- drino and to Elson-Morgan and Molisch reagents, a negative reaction to the reagents of Salcaguchi, Tollens, F'ehling, Millon and Benedict, which contains carbon, hydrogen, oxygen and nitrogen but which does not contain sulfur, halogens or ash, which corresponds to the overall formula C12H25N3O7,

   which forms colorless needle crystals decomposing at 202-205 C, which in 1% aqueous solution exhibits a rotatory power [[alpha]] D21 of more than 95, which does not absorb in the ultraviolet from 220 to 340 milli- microns as shown in Fig. 5 showing the absorbance A on the ordinate and the wavelength on the abscissa and which exhibits characteristic absorption bands in Nujol in the infrared region as shown in FIG. 6 showing the transmission in% on the ordinate and the number of waves on the abscissa. The antibiotic Ni \ -1003 hydrolyzed by heating for 40 minutes at 100 ° C. in solution in hydrochloric acid, loses its antibacterial power.



  The resulting hydrolysis solution was applied as a stain to a ribbon (40 cm x 2 cm) of Toyo No. 50 filter paper, and then developed downward by mixing.

 <Desc / Clms Page number 29>

 butanol-pyridine-acetic acid-water (6: 4: 1: 3 @. The resulting paper chromatogram shows that two spots formed, one of which gave a positive reaction to ninhydrin and the other gave a positive reaction also to ammonia silver nitrate. By comparison with standards, it can be established that the two spots correspond to deoxystreptamine and 6-D-Clucosamine, respectively. For this reason and knowing the molecular weight of the compound, it can be deduced that the antibiotic.



  NK-1003 is a compound consisting of a deoxystreptamine molecule and a 6-D-glucosamine molecule. The antibiotic NK-1003 is substantially non-toxic since its lethal dose when administered intravenously in mice is 1550 mg / kg.



   Antibiotic NK-1003 can be produced by culturing a strain of Streptomyces kanamyceticus in a culture medium which contains a carbohydrate and a nitrogenous alloy, as well as a quantity of antibiotic NK-1001 under aerobic conditions to 'NK-1003 antibiotic accumulates in the culture, then isolating the antibiotic from the culture. The fermentation production of the antibiotic NK-1003 as well as the sources of carbon and nitrogen suitable for this purpose are the same as those suitable for the production of 2'-amino-2'-deoxy- kanamycin. It is preferable to resort to culture or deep immersion with aeration. The culture temperature can be 25 to 30 C but is preferably about 28 C.

   The amount of NK 1001 antibiotic added to the culture medium is not critical, but may be 0.05 to 1.0% and preferably 0.1 to 0.5% by weight of the medium. Antibiotic NK-1001 can be added either at the start of cultivation or during the operation.



   Isolation and purification of the antibiotic NK-1003 can be performed as for 2'-amino-2'-deoxy-kanamycin, as described above.



   In the drawings:

 <Desc / Clms Page number 30>

 Fig. 3 shows the absorption spectrum in the ultraviolet of the antibiotic NK-1001 dissolved in water at 1 mg / ml of water, FIG. 4 represents the absorption spectrum in the infra-
 EMI30.1
 Nit-1001 nuitibiotic wheel in the lqujol; Fig. 5 represents the absorption spectrum in the ultraviolet of the antibiotic NK-1003 dissolved in water at 1 mg / ml of water, Fig. 6 represents the absorption spectrum in the infrared of the antibiotic NK-1003 in Nujol.



   The antibacterial spectra of the antibiotics NK-1001 and NK-1003 which can be used as an additive in the process making the first aspect of the invention are summarized in Table V below.

 <Desc / Clms Page number 31>

 



    TABLE V
 EMI31.1
 Concentration. minîzun olrr 1 inh: l.b i tien en lf \ .p: /; n1 I-licro-orsanisse tried Antibiotic IIK-1001 Antibiotic IX-1003 µr¯atiîs; xe essajYé ± -ntïoiotique i-1 ooi Jmtïbioiique î ± K-? 1, erobacter aerogenes IIà.b 1102 31 2 15 6 A1ca1iEenes faecalis ¯¯¯¯ 3 g Bacillus shelter 7.8. 20 Bacillus STJbtilis ATCC 6633 6,, 25 3,125 Candida albicans> 500> 500 Diplococcus: emior: ise Type 311D 500 250 Escher.cr¯3.a, coli 1; .1 .: 1253 '125 125 K1ebsicll .p: eu. orur¯e 607 31.2 31.2 1, actoba, ci13.us fc: rr. (>! 1ti ATCC 9338 3.9 3.9 Sta1ih.lococcus àiD1% PCI 1200A 15,, 6 7,, 8 StGjîi ' z "IOCOç; C1iS Z1Wet; S 209P. 62.5 62.5 S tï: 1! ilr OCGCCh: '41: 2'F.'1i $ .'E'rc:: l0 Ii: 2 6.25 3 125 I; ca: c¯ctcr ii :. p111 (> 1 :: S 15.6 25;

   CO;: KC ((ri 1. '"6U7 62.5 50 Salmonella' .i..i a.ij'r1F21, A 62.5 62.5 Sa: c., L lut. <: 'A FC' 1 1001 00 250 S, .¯cC'¯a.re ::, ct 'CF2'e:' iS: .Lc '11 -':.: ± 626> 500> 500 ShiGell <:, fi f .'- .3: C2 "; ..? Il 10 2a, 6.25 3 125 8trevt.ocGCCUS lG: iO't '', ¯C'.iS D-90 500 bzz

 <Desc / Clms Page number 32>

 
The process constituting the first aspect of the invention is illustrated with reference to Examples 1 to 8 below.
 EMI32.1
 



  1.?; ;; II ', [; L; Liquid culture medium (15 liters) is inoculated
 EMI32.2
 containing 3.0il starch, 2.01 maltose, 2.01 soy flour, 1.2% sodium nitrate and 0.5% sodium chloride using the typical 0-4- strain 1 of Streptomyces kanamyceticus and incubation is carried out at 28 ° C. with aeration and agitation.



    After 22 hours of incubation, 50 g of 3-amino-glucose are introduced into the culture medium and the incubation is continued.
 EMI32.3
 



  The amount of 2'ino - 2'-deoy-kana:; crre produced in the culture medium reaches its maximum after 150 hours.



   The liquid culture medium is then filtered and the
 EMI32.4
 filtrate using 4 liters of Amber11 te IRC-50 (ammonia form).



  Antibiotics adsorbed to resin are used with 0.5N aqueous auunonia. The active fractions are collected and concentrated to a syrup. One dissolves the syrup in 500 ml of water, then the solution is decolorized by treatment with 6 g of activated carbon. The solution was filtered to remove the activated carbon and the filtrate was subjected to chromatographic elution with 600 µl of Dowex 1x2 (OR form). We elect the fractions
 EMI32.5
 containing 2'-aaino-2'-deoxy-kttnarnycino first before isolating kanamycin A.

   The high concentration fractions were collected and then concentrated and lyophilized to obtain 2.2 g of a crude powder. This crude powder (2.2) is dissolved in 50 ml of water and the solution is treated with
 EMI32.6
 Û'A.13h: llßto CG - 50 (fol'mc auJ.:1lonique). The chromato-; ra .: ucucr, mtzt resin is eluted by means of 0.11-1 0.5N aqueous a.aonialua and the small amount of kananycine is placed 1). Now iuultanu :: nt. The 2'-wimo-2'-dôsoxy- .wll '.. Iycinc is then separated into a single fraction which is concentrated and lyo-

 <Desc / Clms Page number 33>

 philise to get a raw powder.

   By recrystallization of the
 EMI33.1
 powder in a molango of water and diwôt8yLiormamido, 11 do 2'-atrnirto-2'-desoty-ltartarclrxa are obtained under the elm of colorless needle crystals. Elemental analysis indicates that the product contains /.2 ,, 62Î; carbon, 7.52; of iydroune and 1230; J of nitrogen (values found). The molecular weight of the product is determined, which is 435.
 EMI33.2
 : XFJU ... k .. ::.



   A liquid culture medium (15 liters) of the same composition as in Example 1 is inoculated by means of the same
 EMI33.3
 strain of Streptomyces ltunaroyccticus quo in Example 1, xi is carried out incubation at 280C with agitation and aeration. After 22 hours of incubation, 50 g of 6-awinoglucosc are introduced into the culture medium and the incubation is continued, The amount of 2'-amino-2'-deoxy-kanaxvcine accumulating in the "11Ui"} U of culture reaches its maximum at the end of 145 hours after the start of incubation.



   The liquid culture medium is then treated and the crude powder is purified as in Example 1 to obtain 1.0 g.
 EMI33.4
 of crystalline 2-amino-2 '' - deoxy-kanamycin.



  EXE {P.LE, .3 t
Liquid culture medium (15 liters) containing 3.0% starch, 2.5% corn starch, 1.2% sodium nitrate and 0.35% sodium chloride is inoculated using the same strain
 EMI33.5
 of StreptoiVces kanamyceticus than in Example 1, then incubation at 28 ° C. with aeration and agitation is carried out. After 20 hours of incubation, 30 g of the antibiotic NK-1001 are added to the culture medium and the incubation is continued. The production
 EMI33.6
 of 2 '' - amino-2'-deoxy-kanam / cine reaches its maximum after 140 hours of incubation.



   The liquid culture medium is then treated and the
 EMI33.7
 raw powder as in Example 1 to obtain Ze5 g of 2 '' * amino-

 <Desc / Clms Page number 34>

 Crystalline 2'-deoxy-kanamycin.



   A liquid culture medium (15 liters) of the same composition as in Example 3 was inoculated using the same strain
 EMI34.1
 of Stroptonycos kananyocticus than cello of Example 1, then the incubation is carried out at 28 ° C. with aeration and agitation. After 20 hours of incubation, 30 g are added to the culture medium.
 EMI34.2
 a.ntibiotiquo xK-1003 and the incubation is continued. The amount of 2 '' '- amino-2'-desoxy'-kanaMyoine accumulating in the culture medium reaches a maximum after 140 hours of incubation.



   The culture medium is then treated and the crude powder is purified as in Example 1 to obtain 1.5 g of
 EMI34.3
 2's.r:; 3 ... o-2'-desolr-ltariarrdcine. crystalline.



  #: 'ÍPTJE it.:':r
A liquid culture medium (15 liters) having the same composition as in Example 3 is inoculated by means of the
 EMI34.4
 same strain of Streptomyces kanalilyceticus as in Example 1, then the incubation is carried out at 28 ° C. with aeration and agitation.



    After 20 hours of incubation, 45 g of neamine are added to the culture medium and the incubation is continued. The quantity of
 EMI34.5
 2'-amino-2'-desQxy-kanamycin accumulating in the culture medium reaches a maximum after 140 hours of incubation.



   The culture medium is then treated and the crude powder is purified as in Example 1 to obtain 1.2 g of 2'-amino-
 EMI34.6
 2 '-. DLSOxy-itanu :: crystalline ycin.



  EXAMPLE 6. -
A liquid culture medium (15 liters) is sterilized
 EMI34.7
 containing 3.0 starch, ,,, 5 soya farjne, 1.2% sodium nitrate and 0.35% sodium chloride, then cooled and mixed with a sterilized solution and cooled 30 g of antibiotic NK-1001 in water. The mixture is inoculated
 EMI34.8
 resulting in luuyen of the same strain of Streptomyces kananyceticus

 <Desc / Clms Page number 35>

 
 EMI35.1
 than in the example 3 1 and the incubation is carried out at 28C with aeration c3t agitation. The amount of '-ara.no -'- c: eso3-kanu: nyc.nc a; 4Qcutnulant in the culture set reaches a maximum after 140 hours of incubation.
 EMI35.2
 



  The culture medium is treated and the powder is purified
 EMI35.3
 crude as in Example 1 to obtain approximately 2.2 g of 2 '-amino-2'-deoxy-kanamyaInc cr1 p.tall1ne.
 EMI35.4
 EXAMPLE 7, .-
 EMI35.5
 Streptoeces kanawycoticus was grown in the presence of the NI% '-1001 antibiotic as in ex: # 3. The resulting culture medium was filtered and the fold of the i'; iltrt7t (14 13tr) adjusted to 10 per additive. ti on of sodium hydroxide. We ,,, or, net the filtrate then to an extraction by means of Oy25 volume of n-butanol containing 2 g of benzaldsl1yàe per gral1l; llC doantibiotiquos bp-siqueslirdro, soluble contained in the filtrate. We separate lpax-
 EMI35.6
 n-butanolic trait by mixing with 0.1 volume water.

   By adjusting
 EMI35.7
 the pH of the mixture to 2.0 by adding sulfuric acid and stirring,
 EMI35.8
 the water-soluble basic antibiotics are transferred to the aqueous phase. The resulting solution is decolorized with the antibiotic
 EMI35.9
 ques in 2 'water by means of activated carbon and subjected to chromatography on Dowex 1 x 2 resin (forite OH) and chromatography on Amberli te CG-50 resin (f orn! a :: da0-
 EMI35.10
 nique) as in Example 1. A crude powder is obtained which
 EMI35.11
 recrystallized in water and diwetllfo! 1auiQe to obtain 1.1 g of 2'-ar: 1ino-2'-d6soXy-kanamycino under the orao of crystals
 EMI35.12
 colorless needle-like.
 EMI35.13
 



  RX & PLË ¯8..¯-Co! MaraiGon), In this example "a series is carried out to demonstrate the unpredictable and advantageous effect of 7.'Wc: uition of aniinoglucoses and their aerivas.;.: Ystrcptu.- : ini'luC: $ on the formation of the 2'-aaina -'- âéso: yk.na. ^; cinr: CW. "1 the Pl ';: I: ;; tI'1J strong realization of the invention, the copar (.1.ison being established

 <Desc / Clms Page number 36>

 with controls containing no additives.



   For these tests, the typical 0-4-1 strain of Streptomyces kanamyceticus is cultured at 28 ° C. for 150 hours as in Example 11 in identical fermentation tanks containing culture media of the same composition. At various times after the start of the cultivation, as indicated below, various aminoglucoses and their deoxystreptamine compounds are added to the culture media. After continuing the cultivation for 150 hours, the broths were processed as in Example 1 to obtain 2'-amino-2'-deoxy-kanamycin and kanamycin A as far as possible. The quantities of the products obtained and the details concerning the tests are precise in Table VI.

 <Desc / Clms Page number 37>

 
 EMI37.1
 



  B Z E .i U VI
 EMI37.2
 
<tb> Test <SEP> Additive <SEP> Quantity <SEP> Houent <SEP> of <SEP> Content <SEP> in <SEP> Production <SEP> of <SEP> Content <SEP> in <SEP> Production <SEP > of
<tb>
 
 EMI37.3
 1: 't of additive' addition 2 '- <::. Ro :: ino-2'- .ésm:;' {- 2'-amino-2'-deoigi- kanancin A # .nB.ILfcine AT
 EMI37.4
 
<tb> (g) <SEP> after <SEP> the <SEP> kanamycin <SEP> kanarycine <SEP> (mg / ml) <SEP> (g)
<tb> start <SEP> of <SEP> the <SEP> (mg / ml) <SEP> (g)
<tb> put <SEP> in
<tb> culture
<tb> (time)
<tb> 1 <SEP>, <SEP> none <SEP> --- <SEP> - <SEP> 26 <SEP> 0.12 <SEP> 2790 <SEP> 26.8
<tb>
 
 EMI37.5
 2 3-Arsino- 50 22 240 1.10 V180 12.7
 EMI37.6
 
<tb> glucose
<tb> 3 <SEP> 6-Amino- <SEP> 50 <SEP> 22 <SEP> 225 <SEP> 1, <SEP> 00 <SEP> 1730 <SEP> 15.2
<tb> glucose
<tb> 4 <SEP> Antibiotic <SEP> 30 <SEP> 20 <SEP> 575 <SEP> 2.50 <SEP> 1240 <SEP> It,

  2
<tb> NK-1001
<tb> 5 <SEP> same as <SEP> 30 <SEP> 0 <SEP> 480 <SEP> 2.20 <SEP> 1450 <SEP> 13.6
<tb> 6 <SEP> Antibiotic <SEP> 30 <SEP> 20 <SEP> 345 <SEP> 1.50 <SEP> 17.00 <SEP> 16.0
<tb> NK-1003
<tb> 7 <SEP> Néamine <SEP> 45 <SEP> 20 <SEP> 260 <SEP> 1.20 <SEP> 1390 <SEP> 12.8
<tb>
 

 <Desc / Clms Page number 38>

   As can be seen from the results of Table V, the addition
 EMI38.1
 of 1inoGlucoscs and their esoxystreptammic derivatives at mill. ,, '.. 1 of culture leads to a remarkable improvement in the production of 2'-aràino-2'-deoxy-kanazycin, which indicates that the amount of 2e-amino -2e-Deoxy-Aaneunycine produced and accumulated in the culture medium increases greatly when applying the procedure constituting the first aspect of the invention.



   The method constituting the second aspect of the invention is illustrated with reference to Examples 9 to 13,
 EMI38.2
 E.:Ó:-fPrJ 9.- :: A liquid culture medium (15 liters) containing 30; maltose, 2.0% starch, 3.0% soy flour, 1.0% sodium nitrate and 0.5% sodium chloride using mutant A-4-6 from Streptomyces kanamyceticus, then the incubation is carried out at 28 ° C. with aeration and agitation. The quan-
 EMI38.3
 Due 2'-aminu-2'-deo.xy-kanamycin accumulated in the culture mU:! "e1.t reaches a maximum after 6 days after the start of incubation.



   The culture medium is filtered to obtain 14 liters of filtrate which is treated with 4 liters of Amberlite resin.
 EMI38.4
 IRC-50 (aluiaonlue form) The resin is then eluted with 0.5N aqueous ammonia. The active fractions are collected and concentrated into a syrup which is dissolved in 500 ml of water. The solution is decolorized by treatment with 6 g of activated carbon, Or. Filtro the solution to separate the carbon and the filtrate is subjected to a chromatographic elution on 600 ml of Dowex 1 x 2.
 EMI38.5
 (form 011). The 2'-amino-2'-esoxy-kanaiycin is eluted before separating the iianaiivein A.

   The fractions with a high concentration of 2'-D.wino-2'-dú :; iV'-kanúLiJ, Ycine "and then concentrated and lyophilized to obtain 3.2 g of a crude powder. This crude powder is dissolved in 50 ml of water and the solution is treated with Amberlite CG- resin. 50 (ammonia form) to adsorb

 <Desc / Clms Page number 39>

 
 EMI39.1
 antibiotics. We. the resin is subjected to a chroatocraphic 61utlon by means of ar: lr: lOn1aqllc aqueous 0.1 "to 0, 5. A small quantity of loa1ixTycin; is eluted, then the 2'-arnino-2'-deoxy-kan. rcin is eluted in a single fraction.

   This single fraction is concentrated and lyophilized to obtain a crude Par recris- powder.
 EMI39.2
 tallization of this powder in water and ciaétir, yi: 'orna .. ^ ido9 1.9 g of is obtained in the form of colorless needle crystals.
 EMI39.3
 r F.rP. 10-
Liquiao culture medium (15 liters) of the same composition as in Example 9 is inoculated by means of the 3AG mutant
 EMI39.4
 of Streptomyces kanaitqceticus, then one carries out 1) incubation at 280C with aeration and agitation. The amount of 2'-ur: .no-2'-dsoyl <: ana.u1Yc1.'1e sea accumulating in the culture medium reaches a maximum after 6 days after the start of incubation.
 EMI39.5
 



  The ColuLio culture medium is treated in Exenrle 9 to obtain 1.2 g of crystalline 2'-aruino-2'-deoxy-kanarcycin.



  EXAMPLE 11. -
A liquid culture medium (15 liters) having the same composition as in Example 9 is inoculated by means of the mutant
 EMI39.6
 18-8 of Streptomyces kanaàoyceticus, then the incubation is carried out at 280C with agitation and aeration. The amount of 2) -amino-2.1desoxy-kanacrycin accumulating in the culture medium reaches its maximum at 7 days after the start of the escape.



  The culture broth is treated co;> 1; e clans e :: e: 3ple 9 to obtain approximately 2.2 g of 2'-a: aino -? '- déso:; - kana ;; clne cris- talline.



    SIMPLE 12.-
The process of Example 9 is repeated using the mutant
 EMI39.7
 19-2 of Str eptorayces kayiaUceticus. A result analogous to that obtained in Example 9 is obtained. 3.15 g of 2 '-a :: ano- are obtained.

 <Desc / Clms Page number 40>

 
 EMI40.1
 2 '- 1Gsoxy-k :: moun, ycin in crystalline form.



  :; ";: u'F 13, .- (Compare his!
In this example, a Drossais series is carried out to show the advantageous and unpredictable effect of using the mutants A-4-6, 3AG, 18-8 and 19-2 which have a specific aptitude for
 EMI40.2
 The second aspect of the invention is to produce 2-α-Mino-2-dussoxy "kanatnyeinc for the production of 2'-ar> ino-2'-dso> ty-; ianuitycine as the second aspect of the invention. established by culturing the typical 0-4-1 strain of Streptomyces
 EMI40.3
 lçill1.D.: VY ce ticu s.



   For the tests, cultures of the original 0-4-1 strain and A-4-6, 3AG, 18-8 and 19-2 mutants of
 EMI40.4
 St1'81) tol: yces kal1D.h1Ycoticus at 28 C for 150 hours chain in example 9, d; "ns identical fermentation tanks containing a r1: lj \ 1u culture of the composition, At the end of the culture, 2 -. nlno-21-d6soxy-kanamycJne and kanamycin A are assayed in the culture media The results of the tests are summarized in Table or VII below.



    TABLE U VII
 EMI40.5
 Test Souchos used 2'-unino content - kanamycin A content N 2'-dcsoxy-kanamycin (m; /.) ¯ ¯ (m.G / l: l1)
 EMI40.6
 
<tb>
<tb> 1 <SEP> Original <SEP> strain <SEP> 26 <SEP> 2790
<tb> 0--1
<tb>
 
 EMI40.7
 rlutatit A-l-6 430 1970 3, îutant .3AG 285 1785 4 1, iutzJit 1 () - S 520 1900
 EMI40.8
 
<tb>
<tb> 5 <SEP> Mutant <SEP> 19-2 <SEP> 700 <SEP> @ <SEP> 1690
<tb>
 Each of the culture media is treated with a volume of
 EMI40.9
 15 liters cü: .. 1a in the exe, uple 9 to isolate as much as possible 1 ;; 2-sino-2 '' - dusoy-Xanainycin and kana ::: ycin A.

   The quantities 0: .r produi-cs obtained are inaiquécs in Table VIII.

 <Desc / Clms Page number 41>

 T A B L E A U VIII
 EMI41.1
 Test Strains used Proùuct.on: o Production of N 2'-xnLao-2-iiJ5oxj- j (.na.:. Y c.1.no bzz ¯ ¯ kaua: J1ycinc (g) (t;) 1 Oriflinale strain 0 ..12 z6.5
 EMI41.2
 
<tb>
<tb> 0-4-1
<tb>
 
 EMI41.3
 Mutant AIr..6 1..90 19.0
 EMI41.4
 
<tb>
<tb> 3 <SEP> as much <SEP> 3AG <SEP> 1.20 <SEP> 15.8
<tb> 4 <SEP> Mutant <SEP> 18-8 <SEP> 2.20 <SEP> 17.6
<tb> 5 <SEP> Mutant <SEP> 19-2 <SEP> 3.15 <SEP> 16.1
<tb>
 
 EMI41.5
 , ..........., ..............., - .. 11
It emerges from the results in Table VIII that the mutants A-4-6, 3AG ,. 18-8 and 19-2 naturally have 1 ability to produce
 EMI41.6
 2'-amino-2'-àéso> iy-kanai'aycine in a much larger quantity than the gold strain:

  1g '.... nale A-4-1 of Strcptouyccs kill1a.;: Ycoticus.



   Examples 14 and 15 below illustrate the preparation of the antibiotic NK-1001 and the antibiotic NK-1003 which can be used as an additive for carrying out the process constituting the first aspect of the invention.



    EXAMPLE 14.-
Liquid culture medium (15 liters) is inoculated
 EMI41.7
 containing 2.0 ;: starch, 30 c, altose, 3.0,: soy flour., 1.0, sodium nitrate, and 0.5 d of sodium chloride by means of the strain typical 0-4-1 oe Streptorjyccs K <= i1argrecticus and incubation is carried out at 28 ° C. with agitation and aeration. After 80 hours of incubation, 30 L of streptium is added to the medium and the iricubation is continued for a further 80 hours. The amount of IL-1001 antibiotic accumulated in r.d11 or reached its maximum at the end of the incubation period.



  The millou 3rd culture 1lnuide is filtered to obtain 14 disputes this filtrate which is added to. liters of resin An; berlite IRC-50 (faith-rae ammonia) to adsorb antibiotics onto the ion chlrlûGilf. The resin is eluted by means of 0.5N aqueous ammonia, then

 <Desc / Clms Page number 42>

 the active fractions of the eluate are combined and concentrated.



  The concentrate is dissolved in 100 ml of water and the mixture is stirred with 1 g of activated carbon for 30 minutes. by filtration, a discolored solution is obtained which is added to 2 liters of
 EMI42.1
 AMberllto CG-50 plant (strength: aiMuonic), The resin is eluted port .... '1.t los antibiotics adsorbed using an aqueous solution o., i 1 a U, 3a. A single fraction of the i-.00 antibiotic is isolated. firstly after the separation of kanataycin A.



  The single fraction of antibiotic NK-1001 is concentrated and lyophilized to obtain a crude white powder of the antibiotic;: K -1001 (28 J g)
This crude powder is dissolved in 12 ml of water and 50 μl of methanol are added in fractions to deposit the crystals in the solution, After filtration and drying, 17.4 g of the antibiotic NK- are obtained. 1001 in the form of white needle-like crystals.
 EMI42.2
 



  The elcrnental analysis of the product indicates that it contains l1,2 (lJ carbon, 7; 09; J hyclroe; 0ne and 8.95, nitrogen (values found) but no sulfur, d halogens and ash The molecular weight of the product measured by the vapor pressure technique is 492.
 EMI42.3
 1 µl of liquid culture (15 liters) containing 20 d) aj, tidon 3.0-; maltose, 3.0, v soy flour, 1.0; of sodium nitrate and Oe5, sodium chloride by means of the typical strain 0-4-1 of Streptoeiyces kUl1atJy ceticus and the inculi'L.Uil, 28 C is carried out with aeration and agitation.

   After 20 hours of incubation, 25 6 of the NK-1001 antibiotic obtained in QXCIl11J1C 1 are added to this place and the incubation is continued with shaking and 1 ± ion, After 6 days of incubation. , the quantity o. ' r..ntibiotiq1.w Nîi-1003 a4cuß: ule in culture reaches its maximum.

 <Desc / Clms Page number 43>

 The culture medium is filtered to obtain 14 liters
 EMI43.1
 of a filtrate which was treated with 4 liters of Koycn Aborlito Iiic-50 (aJ form: 111lonic) to adsorb the antibiotics xon the resin. The resin is then eluted with aqueous ci (-, la!. 'J.i0ni! .Quo 0, sil and the active fractions are combined and diluted and concentrated.
 EMI43.2
 Dissolve the concentrate in 100 l of water and add 1 g of activated carbon.

   The resulting decolored solution is filtered and treated with 2 liters of Ailberl3.te CG-0 resin (a.,., U.0nic bore). The resin bearing the adoI'b6s antibiotics was washed with water, then eluted with 0.3X aqueous a: uon.1qua to separate the kanatycin A fraction first and then a single fraction of kanatycin A. the antibiotic NK-1003. This single fraction of the NK-1003 antibiotic was concentrated and lyophilized to obtain 11.3 g of this NK-1003 antibiotic as a crude powder. By recrystallization, powder in water and
 EMI43.3
 methanol, then drying, 7.5 g of tib: 1, otlque NK-1003 are obtained in the form of white needle crystals.
 EMI43.4
 



  Elbiaental analysis of the product indicates that it contains,. ,, 2; of carbon, 7, 81 dhydrocne and l; "11; nitrogen (values found). The molecular weight of the product, 4 tninL by the vapor pressure technique is 305 (the product contains a methanol molecule of crystallization ).

 

Claims (1)

EMI44.1 CLAIMS EMI44.2 1.- Produces ue the 2'-aiàino-2'-dexoxylLanax;, ycine admits an increased roundness characterized in co that we cultivate a strain of tx'cSptouyces .K: '. 1la.::rct1cus in aes cvnui tions aÓx'obiefs in a normal culture medium contained a carbohydrate and a nutrient nitrogenous and in the presence of'4U less one uaaitif chosen by, nor the arninoalucosos and their glucosidiquos compounds combined with the degoj.ystre) ttlfl. : ine, until the '-àino .'- dsoxy-iia..an, ycin4 accumulates in substantial amounts in the culture medium, after which the 2'-szino-2'dso: xy is collected -lté1namycin from the culture medium. 2.- A method according to claim 1. characterized in that 1'awiiioiluotose added is 3-aninoùlucose or 6-amt-no- (T, .1; = CâOv EMI44.3 3.- Method according to claim 1, characterized in EMI44.4 co that the compound; 7.ucos: iâic of a; .1inoglucose combined with d ::; o: - :: ystrvpt ± ll: 1inc added is chosen from pÍU'olilaIlline, deoxy-: rcptuirm.0 -icyiosaniniâe, nci, .. mine .. the antibiotic Nri-10Q1 and the antibiotic NK-1003. 4.- Cie production process of '-aaino-2'-àesoxylC: "\ 1l, I" yc1no with an increased rondomont characterized in that one cultivates a.:, Utr..L1t lotO ûtrc, toïces ltanwivceticus which has an ability n ;;. i, Ji'eàài 1, pronounce âav.n;, e 0.13 2'-inino-2'-déscxy-klilia ùycin 4L (;:, cE: \ Ol) LG :: vrtivn; â aorobis in the usual Cit3 culture medium CC? bd.t'.T'l, ü2ij les:., 'G4ll'C?: ß usual ae carbon and nitrogen up to c, cu, :: ul ;. ian <; e la 21- 3Ji.inl, j-2 "-dvso,: ry-kwlD.ycin.;. in a quantity; izl, 1 ;,., <; nt 1,: ,, Jarii,; iie (IN the middle..pu1s the 2'-aminor; '^ tsta;: ilirl .., ^. i .E's3 .... 1.1 culture medium. 3.- 'OI.: Uj uo tJiVlut: vUl1 de la 2' - # àino-2> -déso; j - :: ':' 11 ".... 'dJ, 0 ü,' VLC: un :, in, 1 <;;., accl'u earacturise in CE2 that we cul- ...: V ...: le û.vtwil4 A -.- o, 3Au, 1d-8 or 19-2 da Streptouyces 1, ... nc: u; .yce .. <Desc / Clms Page number 45> ticus under aerobic conditions in a usual culture medium containing a carbohydrate and a nitrogenous nutrient until accumulation of 2'-amino-2'-deoxy-kanamycin in a usefully large quantity in the culture medium and then collected. 2'-amino-2'-deoxy-kanamycin from the culture medium, 6. A method according to any one of the preceding claims, characterized in that the 2'-amino-2'-deoxy-kanamycin is collected from the culture medium by treating it with a cation exchange resin which adsorbs antibiotics, after which the resin is eluted, treated the eluate by means of an anion exchange resin, this resin is eluted, the eluate is treated again with a cation exchange resin and the resin is subjected to chromatographic elution by means of aqueous ammonia . 7. A process according to any one of the preceding claims, characterized in that the 2'-amino-2'-deoxy-kanamycin is collected from the culture medium by filtering the latter, by adjusting the pH of the resulting filtrate. to 10 by addition of an alkali metal hydroxide, subjecting the filtrate to extraction with n-butanol containing a certain amount of benzaldehyde, mixing the resulting extract with water, adjusting the pH of the mixture of 2 by addition of sulfuric acid, stirring this mixture to transfer the water-soluble basic antibiotics into the aqueous phase, then separating the aqueous phase and subjecting it to chromatography on an anion exchange resin with subsequent elution, followed by carrying out a new chromatography with a cation exchange resin and a new elution.