Abstract:
Disclosed and claimed is an improved fermentation process for preparing the known antibiotic U-43,120, herein referred to as paulomycin. Also disclosed and claimed are the novel and useful antibiotics paulomycin A and paulomycin B.

Description:
BACKGROUND OF THE INVENTION 
     The production of antibiotic U-43,120 by Streptomyces paulus, NRRL 8115, and the physicochemical properties of this compound are described in U.S. Pat. No. 3,988,441 (Oct. 26, 1976). Recent work by us shows, unexpectedly, that antibiotic U-43,120, which we have now named paulomycin, is a mixture of two chemically related antibiotics. We have named these two new antibiotic entities paulomycin A and paulomycin B. 
     BRIEF SUMMARY OF THE INVENTION 
     An improved fermentation process for preparing paulomycin has been discovered. This process uses a biologically pure culture of a newly-discovered strain of S. paulus. This new man-made culture has been designated S. paulus strain 273. 
     We have also discovered, unexpectedly, that paulomycin is a mixture of two chemically related antibiotics. These new and useful antibiotics have been named paulomycin A and paulomycin B. 
     Paulomycin A and B have the properties of adversely affecting the growth of Gram-positive bacteria, for example Bacillus subtilis, Staphylococcus aureus, Streptococcus pyogenes and Streptococcus faecalis. Thus, they can be used alone or in combination with other antibacterial agents to prevent the growth of, or reduce the number of, such microorganisms present in various environments. Also, they are useful in wash solutions for sanitation purposes, as in the washing of hands and in the cleaning of equipment, floors, or furnishings of contaminated rooms or laboratories; they are also useful as an industrial preservative, for example, as a bacteriostatic rinse for laundered clothes and for impregnating papers and fabrics; and they are useful for suppressing the growth of sensitive organisms in plate assays and other microbiological media. 
     DETAILED DESCRIPTION OF THE INVENTION 
     Chemical and Physical Properties of Paulomycin A 
     Infrared Absorption Spectrum 
     Paulomycin A has a characteristic infrared absorption spectrum in a mineral oil mull as shown in FIG. 1 of the drawings. Peaks are observed at the following wave lengths: 
     
         ______________________________________Band                   BandFrequency.sup.1       Intensity.sup.2                  Frequency   Intensity______________________________________3356        40         1241        203266        51         1187        403230        51         1153        293072        74         1138        222954         4         1118        242923         2         1097        252870        12         1055        292854         5         1026        212724        75         990         322672        76         940         652248        85         931         632050        29         910         461735         7         894         601695        26         868         671640        42, sh.    834         711626        31         818         611579        24         785         661491        47, sh.    751         411457        12         723         581378        19         703         601340        39         665         531296        251261        15______________________________________ .sup.1 Wavenumbers (cm.sup.-1) .sup.2 Percent transmittance (% T), sh. = Intensity at 3800 cm.sup.-1 is 84.9% T. Minimum intensity at 1855 cm.sup.-1 is 88.7% T. 
    
     Paulomycin A also has a characteristic infrared absorption spectrum when pressed in a KBr pellet. Peaks are observed at the following wave lengths: 
     
         ______________________________________Band                 BandFrequency.sup.1       Intensity.sup.2                Frequency    Intensity______________________________________3467        34, sh.  1153         17, sh.3421        29       1137         133270        37       1117         143236        39       1098         153072        63       1055         172946        35       1026         112880        49       991          212831        59       943          542736        65       931          542245        69       909          342047        19       897          45, sh.1735         2       869          531701        15       834          601640        28, sh.  816          511625        22       785          571578        14       751          371490        37, sh.  728          581454        16       702          57, sh.1384        15       690          551376        18, sh.  666          521340        30       639          551296        16       624          551261         9       603          531244        12, sh.  527          581188        27       487          47______________________________________ .sup.1 Wavenumbers (cm.sup.-1) .sup.2 Percent transmittance (% T), sh. = Intensity at 3800 cm.sup.-1 69.9% T Minimum intensity at 1905 cm.sup.-1 is 72.8% T 
    
     Ultraviolet Absorption Spectrum 
     The UV spectrum of paulomycin A is shown in FIG. 2 of the drawings. The solution of antibiotic in 95% ethanol displayed absorption as follows: 
     
         ______________________________________   λ max         (a)______________________________________   236 nm         (20.57)   276   (13.76)   322   (12.33)______________________________________ 
    
     Proton Magnetic Resonance ( 1  H-NMR)Spectrum 
     The  1  -H-NMR spectrum of paulomycin A at 100 MHZ is shown in FIG. 3 of the drawings. The  1  H-NMR spectrum was observed on a Varian XL-100-15 Spectrometer on a solution (ca. 0.5 ml., ca. 150  mg./ml.) of the sample of the antibiotic in deutero-dimethylsulfoxide (d 6  -DMSO). The spectrum was calibrated against internal tetramethylsilane and frequencies were recorded in ppm downfield from tetramethylsilane. 
     Melting Point: 95°-105° C. 
     Optical Rotation: [α] D   25 , +27° (c, 0.9435, CHCl 3 ) [α] D   25 , -22° (c, 0.9555, methanol). 
     Titration Data: pKa, 7.4; Eq.wt. 808 Solvent, 60% aq. EtoH; titrant, KOH 
     Elemental Analysis: C, 48.77; H, 5.73; N, 3.29; S, 4.39; O (by difference), 37.82. Calcd. for C 34  H 46  N 2  SO 17  : C, 51.90; H, 5.85; N, 3.56; S, 4.07; O (by difference), 34.60. 
     Solubilities: Highly soluble in methanol, ethanol and other alcohols, acetone and other ketones, ethyl acetate and other esters, chloroform, methylene chloride, acetonitrile, DMF, DMSO, and the like. Relatively insoluble in saturated hydrocarbon solvents and water. 
     Electrophorectic Anaylsis: pH 1.8, +0.14; pH 8.7, -2.30. 
     Biological Properties of Paulomycin A 
     Antitumor (in vitro) Activity: L-1210; ID 50 , 3.4 mcg./ml.; ID 90 , 9.0 mcg./ml. 
     Antimicrobial Spectrum of Paulomycin A 
     Paulomycin A is active against various Gram-positive bacteria as shown in the following table: 
     Assay 
     The antibacterial assay is a standard microplate agar assay using PYG agar, pH 6. PYG agar consists of the following ingredients: 
     
         ______________________________________Peptone                10    g./l.Yeast extract          5     g./l.Glucose                1     g./l.Agar                   15    g./l.Distilled water, q.s.  1     l.______________________________________ 
    
     The MIC is determined by standard methods. The inocula are overnight cultures of the test organisms, diluted so that the final population contains approximately 10 5  cells/ml. The agar plates are incubated at 28° to 37° C. for 24 hours. The lowest antibiotic concentration which allows no growth=MIC or minimum inhibitory concentration. 
     
         ______________________________________           Minimum Inhibitory ConcentrationOrganism        (mcg/ml)______________________________________S. aureus  UC 76    0.125      6685     0.25      6690     0.25S. pyogenes      152S. pneumoniae      41S. faecalis      694      15.6Enterococcus sp.      701      1.0E. coli    45       125K. pneumoniae      58       125Ps. aeruginosa      95       125S. schottmuelleri      126      125S. marcescens      131      62.5S. flexneri      143      62.5P. stuartii      6570     62.5E. cloacae 6783     250______________________________________ 
    
     &#34;UC&#34; is a registered trademark of the Upjohn Company Culture Collection. These cultures can be obtained from the Upjohn Company in Kalamazoo, Michigan, upon request. 
     Chemical and Physical Properties of Paulomycin B 
     Infrared Absorption Spectrum 
     Paulomycin B has a characteristic infrared absorption spectrum in a mineral oil mull as shown in FIG. 4 of the drawings. Peaks are observed at the following wave lengths: 
     
         ______________________________________Band                 BandFrequency.sup.1       Intensity.sup.2                Frequency    Intensity______________________________________3365        38       1157         183267        46       1135         173233        47       1118         182953         2       1098         182923         0       1065         23, sh.2868         6, sh.  1056         222854         2       1026         162727        75       996          27, sh.2674        77       992          272248        90       944          602047        27       929          571735         5       910          441696        20       897          55, sh.1627        27       869          651518        20       855          641457         7       839          691377        13       817          611342        32       784          671297        22       751          491261        11       721          551243        18, sh.  690          601197        34       664          55______________________________________ .sup.1 Wavenumbers (cm.sup.-1) .sup.2 Percent transmittance (% T), sh. = Intensity at 3800 cm.sup.-1 is 89.8% Minimum intensity at 1846 cm.sup.-1 is 93.6% T. 
    
     Paulomycin B also has a characteristic infrared absorption spectrum when pressed in a KBr pellet. Peaks are observed at the following wave lengths: 
     
         ______________________________________Band                 BandFrequency.sup.1       Intensity.sup.2                Frequency    Intensity______________________________________3473        34, sh.  1157         143419        28       1136         133390        29, sh.  1118         133273        37       1099         143240        39, sh.  1055         163075        69       1026         112941        38       995          22, sh.2831        59       991          212729        65       943          532238        70, sh.  928          502046        20       909          361735         2       897          46, sh.1700        14       869          531638        28, sh.  855          531626        22       840          58, sh.1578        15       816          521490        35, sh.  784          581450        17       751          431385        16       729          591372        18       702          58, sh.1341        28       691          551296        18       666          541261         8       624          551243        13, sh.  602          531195        28       490          46______________________________________ .sup.1 Wavenumbers (cm.sup.-1) .sup.2 Percent transmittance (% T), sh. = Intensity at 3800 cm.sup.-1 is 70.2% Minimum intensity at 1911 cm.sup.-1 is 74.0% T. 
    
     Ultraviolet Absorption Spectrum 
     The UV spectrum of paulomycin B is shown in FIG. 5 of the drawings. The solution of antibiotic in 95% ethanol displayed absorption as follows: 
     
         ______________________________________   λ max         (a)______________________________________   236 nm         (20.29)   276   (13.51)   322   (11.97)______________________________________ 
    
     Proton Magnetic Resonance ( 1  H-NMR) Spectrum 
     The  1  -H-NMR spectrum of paulomycin B at 100 MHZ is shown in FIG. 6 of the drawings. The  1  H-NMR spectrum was observed on a Varian XL-100-15 Spectrometer on a solution (ca. 0.5 ml., ca. 150 mg./ml.) of the sample of the antibiotic in deutero-dimethylsulfoxide (d 6  -DMSO). The spectrum was calibrated against internal tetramethylsilane and frequencies were recorded in ppm downfield from tetramethylsilane. 
     Melting Point: 105.6°-143° C. with decomposition. 
     Optical Rotation: [α] D   25 , +19° (c, 0.798, CHCl 3 ) [α] d   25 , -28° (c, 0.850, methanol) 
     Titration Data: pKa, 7.4; Eq.wt., 791. Solvent: 60% ethanol; titrant, KOH. 
     Elemental Analysis: C, 50.82; H, 5.71; N, 3.51; S, 4.14; O (by difference) 35.82. Calcd for C 33  H 44  N 2  SO 17  ; C, 51.29; H, 5.69; N, 3.62; S, 4.14; O (by difference) 35.23. 
     Solubilities: Highly soluble in methanol, ethanol and other alcohols, acetone and other ketones, ethyl acetate and other esters, chloroform, methylene chloride, acetonitrile, DMF, DMSO, and the like. Relatively insoluble in saturated hydrocarbon solvents and water. 
     Electrophorectic Analysis: pH 1.8+0.15; pH B.7 -0.92. 
     Biological Properties of Paulomycin B 
     Antitumor (in vitro) Activity: L-1210; ID 50 , 8.6 mcg./ml.; ID 90 , 21 mcg./ml. 
     Antimicrobial Spectrum of Paulomycin B 
     Paulomycin B is active against various Gram-positive bacteria as shown in the following table. 
     Assay. The antibacterial assay is a standard microplate agar assay using PYG agar, pH 6. The MIC is determined by standard methods. The inocula are overnight cultures of the test organisms, diluted so that the final population contains approximately 10 5  cells/ml. The agar plates are incubated at 28° to 37° C. for 24 hours. The lowest antibiotic concentration which allows no growth=MIC or minimum inhibitory concentration. 
     
         ______________________________________           Minimum Inhibitory ConcentrationOrganism        (mcg/ml)______________________________________S. aureus  UC 76    0.50      6685     0.50      6690     2.0S. pyogenes      152S. pneumoniae      41S. faecalis      694      125Enterococus sp.      701      31.2E. coli    45       250K. pneumoniae      58       250Ps. aeruginosa      95       250S. schottmuelleri      126      500S. marcescens      131      250S. flexneri      143      250P. stuartii      6570     250E. cloacae 6783     500______________________________________ 
    
     Comparison of paulomycin A and B on thin layer chromatography is as shown in FIG. 7 of the drawings. 
    
    
     Reference to the Drawings 
     FIG. 1: Infrared Absorption Spectrum of Paulomycin A in a Mineral Oil Mull. 
     FIG. 2: Ultraviolet Absorption Spectrum of Paulomycin A. 
     FIG. 3: Proton Magnetic Resonance Spectrum of Paulomycin A. 
     FIG. 4: Infrared Absorption Spectrum of Paulomycin B in a Mineral Oil Mull. 
     FIG. 5: Ultraviolet Absorption Spectrum of Paulomycin B. 
     FIG. 6: Proton Magnetic Resonance Spectrum of Paulomycin B. 
     FIG. 7: Thin layer Chromatographic Comparison of Paulomycin A and B. 
    
    
     THE MICROORGANISM 
     The man-made, biologically pure culture of the newly-discovered strain of S. paulus useful in the subject invention proces is S. paulus, strain 273, NRRL 12251. 
     A subculture of this microorganism can be obtained from the permanent collection of the Northern Regional Research Laboratory, U.S. Department of Agriculture, Peoria, Ill., U.S.A. Its accession number in this depository is NRRL12251. It should be understood that the availability of the culture does not constitute a license to practice the subject invention in derogation of patent rights granted with the subject instrument by governmental action. 
     The microorganism of this invention was studied and characterized by Alma Dietz and Grace P. Li of The Upjohn Research Laboratories. 
     S. paulus strain 273, NRRL 12251, can only be differentiated from S. paulus, NRRL 8115 by the ability of S. paulus strain 273 to produce higher fermentation titers of antibiotic U-43,120. As disclosed above, this antibiotic has been found to be a mixture of paulomycin A and B. It is because of this fermentation production difference that the microbe of this invention is considered to be a strain of S. paulus, NRRL 8115. 
     The taxonomy of S. paulus, NRRL 8115 is disclosed in U.S. Pat. No. 3,988,441. The same taxonomy can be used to describe S. paulus strain 273, NRRL 12251. 
     The compound of the invention process is produced when the elaborating organism is grown in an aqueous nutrient medium under submerged aerobic conditions. It is to be understood, also, that for the preparation of limited amounts, surface cultures and bottles can be employed. The organism is grown in a nutrient medium containing a carbon source, for example, an assimilable carbohydrate, and a nitrogen source, for example, an assimilable nitrogen compound or proteinaceous material. Preferred carbon sources include glucose, brown sugar, sucrose, glycerol, starch, cornstarch, lactose, dextrin, molasses, and the like. Preferred nitrogen sources include cornsteep liquor, yeast, autolyzed brewer&#39;s yeast with milk solids, soybean meal, cottonseed meal, cornmeal, milk solids, pancreatic digest of casein, fish meal, distillers&#39; solids, animal peptone liquors, meat and bone scraps, and the like. Combinations of these carbon and nitrogen sources can be used advantageously. Trace metals, for example, zinc, magnesium, manganese, cobalt, iron, and the like, need not be added to the fermentation media since tap water and unpurified ingredients are used as components of the medium prior to sterilization of the medium. 
     Production of the compound by the invention process can be effected at any temperature conducive to satisfactory growth of the microorganism, for example, between about 18° and 40° C., and preferably between about 20° and 28° C. Ordinarily, optimum production of the compound is obtained in about 3 to 15 days. The medium normally remains acid during the fermentation. The final pH is dependent, in part, on the buffers present, if any, and in part on the initial pH of the culture medium. 
     When growth is carried out in large vessels and tanks, it is preferable to use the vegetative form, rather than the spore form, of the microorganism for inoculation to avoid a pronounced lag in the production of the compound and the attendant inefficient utilization of the equipment. Accordingly, it is desirable to produce a vegetative inoculum in a nutrient broth culture by inoculating this broth culture with an aliquot from a soil, liquid N 2  agar plug, or a slant culture. When a young, active vegetative inoculum has thus been secured, it is transferred aseptically to large vessels or tanks. The medium in which the vegetative inoculum is produced can be the same as, or different from, that utilized for the production of the compound, so long as a good growth of the microorganism is obtained. 
     A variety of procedures can be employed in the isolation and purification of the compound of the subject invention from fermentation beers, for example, solvent extraction, partition chromatography, silica gel chromatography, liquid-liquid distribution in a Craig apparatus, adsorption on resins, and crystallization from solvents. 
     In a preferred recovery process the compound of the subject invention is recovered from its culture medium by separation of the mycelia and undissolved solids by conventional means, such as by filtration or centrifugation. The antibiotic is then recovered from the filtered or centrifuged broth by extraction. For the extraction of paulomycin from the filtered broth, water-immiscible organic solvents in which it is soluble, for example, chloroform, ethylene dichloride, ethyl acetate, and methylene chloride (preferred) can be used. Advantageously, the extraction is carried on after the filtered beer is adjusted to a pH of about 2 to 7 with a mineral acid. The methylene chloride extracts are combined and evaporated to dryness under vacuum. 
     The first step in purification of the methylene chloride extract, as described above, is the use of silica gel chromatography using as solvents ethyl acetate, ethyl acetate-ethanol-water, ethyl acetate-water, and chloroform-ethyl acetate. The active fractions from the silica gel column can be further purified by countercurrent distribution using cyclohexane-ethyl acetate, 95% ethanol-water (1:1:1:1) as the solvent system. Fractions from the countercurrent distribution can be subjected to further silica gel chromatography to obtain a more active preparation which is thus more pure. A final purification step to yield a pure crystalline product is conducted by using chloroform to crystallize the product from the silica gel chromatography. 
     Preparations of paulomycin can be purified by repeated silica gel chromatography, as described above, without resort to countercurrent distribution. 
     Paulomycin A can be isolated as an essentially pure crystalline preparation from paulomycin by use of partition chromatography using a solvent system consisting of dioxane-cyclohexane, pH 7.0-0.1 M phosphate buffer (35:65:8). 
     Paulomycin B can be isolated as an essentially pure crystalline preparation from paulomycin by countercurrent distribution using a solvent system consisting of acetone-ethyl acetate-hexane, pH 7.0-0.1 M phosphate buffer (6:1:6:2 v/v). 
     Salts of paulomycin A and B can be formed with inorganic cations, for example, sodium, potassium, lithium, and calcium, since both antibiotics are weakly acidic. Such salts can be prepared, as for example, by suspending the antibiotic in water, adding a dilute base until the pH of the solution is about 10.0 to 11.0, and freeze-drying the solution to provide a dried residue consisting of the paulomycin A or B salt. Salts of paulomycin A or B can be used for the same biological purposes as the parent compounds. 
     Hereinafter are described non-limiting examples of the process and products of the subject invention. All percentages are by weight, and all solvent mixture proportions are by volume unless otherwise noted. 
     EXAMPLE 1 
     A. Fermentation 
     A biologically pure culture of Streptomyces paulus strain 273, NRRL 12251, is used to inoculate a series of 500-ml. Erlenmeyer seed flasks, each containing 100 ml. of sterile seed medium consisting of the following ingredients: 
     
         ______________________________________Glucose monohydrate     25 g/lPharmamedia*            25 g/lTap water q.s.           1 liter______________________________________ *Pharmamedia is an industrial grade of cottonseed flour produced by Traders Oil Mill Company, Fort Worth, Texas. 
    
     The seed medium presterilization pH is 7.2. The seed inoculum is grown for 2 days at 28° C. on a Gump rotary shaker operating at 250 r.p.m. and having a 21/2 inch stroke. 
     Seed inoculum (5%), prepared as described above, is used to inoculate a series of 500 ml. fermentation flasks containing 100 ml. of sterile fermentation medium consisting of the following ingredients: 
     
         ______________________________________Glucose monohydrate     22 g/lPharmamedia             19 g/lDextrin                 20 g/lBrewers yeast           0-1 g/lUcon (defoamer)         10 g/lTap water q.s.           1 liter______________________________________ 
    
     Note: pH was adjusted to 7.2 before sterilization. 
     The inoculated fermentation flasks are incubated at a temperature of 25° C. for 3 to 5 days while being shaken on a Gump rotary shaker operating at 250 r.p.m. and having a 21/2 inch stroke. Foaming in the fermentation flasks is controlled by the antifoam agent UCON (a synthetic defoamer supplied by Union Carbide, N.Y., N.Y.) 
     A comparison of the paulomycin fermentation titers of S. paulus, NRRL 8115 and S. paulus strain 273, NRRL 12251 under identical fermentation conditions is as follows: 
     
         ______________________________________  Anti-S. lutea (pH 6.0 phosphate buffer, 32° C.)  Biounits per ml.Culture  Day 1    Day 2    Day 3  Day 4  Day 5______________________________________NRRL     42       51       64     51     32NRRL 8115    &lt;1       3        &lt;1     &lt;1     8______________________________________ 
    
     The above results clearly show the superiority of S. paulus strain 273, NRRL 12251, in the fermentation production of paulomycin. 
     Antibiotic Assay 
     Fermentation beers are sedimented by centrifugation at ca. 3000×g. The supernatant fluids (clear beers) are assayed for antibiotic activity v.s. S. lutea, UC®130 using bioautographic or disc-plate methods. For bioautography, thin layer chromatography (tlc) is performed on Brinkman silica gel (Sil NHr plates) or on Brinkman cellulose (Cel 400 ) developed respectively in CHCl 3 , C 2  H 5  OH and H 2  O (25:30:5) or in 0.1 M potassium phosphate, pH 7. Clear beer anti-S. lutea biounit titers are obtained by a standard disc-plate assay. 
     A biounit (BU) is defined as the concentration of the antibiotic which gives a 20 mm zone of inhibition under the above assay conditions. Thus, if for example a fermentation beer, or other solution containing the antibiotic, needs to be diluted 1/100 to give a 20 mm zone of inhibition, the potency of such beer or solution is 100 BU per ml. 
     B. Recovery 
     (1) Extraction 
     Whole beer, ca 4000 liters, obtained from a fermentation as described above, is adjusted to pH 2.8 with sulfuric acid and cooled to 16° C. in the fermentor. Celatom FW-40 filter aid (The Eagle Pitcher Co., Cincinnati, Ohio), 237 kg. is added and the mixture is filtered using a rotary vacuum filter. The filtered beer which contains 9% of the total bioactivity is discarded. The filter cake is slurried with 2000 liters ethyl acetate overnight then filtered through 40 chambers of a 30&#34; press filter. The filtrate (ethyl acetate extract containing paulomycin) is concentrated to 64 liters. This concentrate is mixed with 17.5 kg. of Harborlite 2000 M (Harborlite Corporation, Escondido, Cal.) and the wet mixture was dried in a vacuum overnight at 40° C. The dried Harborlite-paulomycin mixture is used as described below: 
     (2) Purification - Harborlite Leaching Column Chromatography 
     The dried harborlite-paulomycin mixture, obtained as described above, is loaded into a 14&#34; column partially filled with dried heptane and packed under atmospheric pressure. The column is eluted at a rate of 1.5 liter per minute and fractions (as shown below) are collected. 
     
         ______________________________________                              Percent of                              BioactivityFraction                  Volume   of StartingNumber Eluting Solvent    (1)      Material______________________________________1      Heptane            107      42      Heptane-Ethyl acetate (97:3)                     103      13      Heptane-Ethyl acetate (94:6)                     93       14      Heptane-Ethyl acetate (91:9)                     93       35      Heptane-Ethyl acetate (88:12)                     93       76      Heptane-Ethyl acetate (80:15)                     93       157      Heptane-Ethyl acetate (80:15)                     93       178      Heptane-Ethyl acetate (80:15)                     93       139      Heptane-Ethyl acetate (80:15)                     93       1710     Heptane-Ethyl acetate (80:15)                     93       1511     Heptane-Ethyl acetate (80:15)                     93       1112     Heptane-Ethyl acetate (80:15)                     93       1013     Heptane-Ethyl acetate (80:15)                     93       314     Heptane-Ethyl acetate (80:15)                     93       115     Heptane-Ethyl acetate (80:15)                     93       &lt;1______________________________________ Fractions are pooled as follows: Pool A, Fractions 10 and 11. Part B, Fractions 7, 8, and 12. Part C, Fractions 6, 9, 13 and 14. Part D, Fractions 5 and 15. 
    
     The pools are concentrated to dryness in vacuo. Crystalline paulomycin is obtained from pools A, B and C by the following procedures. 
     (3) Crystallization of paulomycin 
     The residue obtained from pool A contains ca 44 g. of paulomycin. This material is dissolved in 4.2 l. of methylene chloride. Heptane, 4.2 l., is added under stirring. An oily material precipitates and is removed by decantation. The oil is dissolved in acetone and added to the supernatant. Then, an additional 8.4 liter of heptane is added and the mixture is stored at 5° C. overnight. Crystalline material precipitated is isolated by filtration and dried [Prep. A, 47.7 g., containing ca 29.7 g. of paulomycin]. The filtrate which contains ca 7.9 g. of paulomycin is concentrated to a small volume (ca 3 liter) and is stored for further processing and isolating the remaining paulomycin. 
     The residue from pool B is treated as described above to give Preparation B, 56.1 g. of crystals containing ca 43.1 g. of paulomycin. The mother liquors contain ca 3.8 g. of paulomycin. 
     The residue from pool C is treated as described for pool A to give Preparation C, 70.5 g. of crystals containing ca 35.1 g. of paulomycin. The mother liquors contain 7.6 g. of paulomycin. 
     (4) Combination of Crystalline Preparations Recrystallization 
     Preparations, A, B and C are combined to give prep. 64.1, 174.3 g. 
     Preparation 64.1 is dissolved in chloroform (37.5 ml./g. of preparation 64.1) by heating on a steam bath. The solution is clarified by filtration and the clear solution is mixed with ether (18.75 ml. per g. of -64.1) and heptane (18.75 ml. per g. of -64.1) under heating on a steam bath. The slightly cloudy solution is allowed to stand at room temperature for 2 hours then at 5° C. for 20 hours. Crystalline paulomycin is isolated by filtration (prep. -64.2, 110.61 g.). The mother liquors give prep. -64.3, 60 g. by concentration to dryness in vacuo. 
     Crystalline paulomycin (prep -64.2), isolated as described above, is recrystallized from chloroform-ether-heptane as described above to give preparation -65.1, 86.13 g. The new mother liquors give preparation -65.2. Preparation -65.1 contains 66.8% paulomycin A and 32.9% paulomycin B as determined by HPLC. 
     EXAMPLE 2 
     Paulomycin A-Isolation by Partition Chromatography 
     Four hundred g. of dicalite-diatomite is slurried with upper phase of the system consisting of dioxane-cyclohexane-pH 7.0-0.1 M phosphate buffer (35:65:8). Lower phase, 160 ml., is added and the whole is mixed for 20 minutes. The slurry is added into a column and packed to a constant height under 3 Atm. pressure. 
     The starting material 1 g. of crude crystalline paulomycin, obtained as described above, is dissolved in 5 ml. of lower phase and 15 ml. of upper phase, mixed with 15 g. of dicalite, and added on the top of the column. The column is eluted with the upper phase. Fractions of 10 ml. are collected at a rate of 4 ml./min. The chromatography is followed by testing for bioactivity vs. S. lutea and by tlc (Brinkman&#39;s Cellulose 400; pH 7.0 phosphate buffer). Fractions containing paulomycin A are combined and concentrated to dryness. The residue is crystallized from 5 ml. of chloroform and 5 ml. of ether to give 120 mg. of an essentially pure crystalline preparation of paulomycin A. 
     EXAMPLE 3 
     Paulomycin B-Isolation by Countercurrent Distribution 
     Solvent: acetone-ethyl acetate-hexane-pH 7.0-0.1 M phosphate buffer (6:1:6:2 v/v). 
     Crude crystalline paulomycin, 3.0 g., is dissolved in both phases of the solvent system and introduced in 4 tubes of an all-glass 500-tube countercurrent distribution apparatus. After 800 transfers, the distribution is analyzed by testing for bioactivity vs. Sarcina lutea and by tlc (Brinkman&#39;s Cellulose 400, pH 7.0 phosphate buffer). Fractions containing paulomycin B only (fraction 210-273) are combined and the solution is adjusted to pH 5.5 with 1 N aqueous hydrochloric acid. The lower phase is extracted 3 times with 1/3 ethyl acetate volume each time. The ethyl acetate extracts are combined with the upper phase and the combined solution is concentrated to dryness in vacuo. The residue is crystallized from 15 ml. chloroform, 7.5 ml. of ether and 7.5 ml. of heptane to give 290 mg. of an essentially pure crystalline preparation of paulomycin B. Recrystallization from the same solvents yielded 170 mg. of an essentially pure crystalline preparation of paulomycin B.