• 专利附录
  • 专利说明
  • 权利要求
  • 类似技术
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  • 引用文献
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  • 优先权
    • 专利摘要:
    A process for preparing tylactone (20-dihydro-20,23- dideoxytylonolide), which has the formula: by submerged aerobic fermentation of Streptomyces fradiae NRRL 12188 or a tylactone-producing mutant or recombinant thereof.
    公开号:SU1069631A3
    申请号:SU813302650
    申请日:1981-06-30
    公开日:1984-01-23
    发明作者:Генри Балтз Ричард；Томас Сено Юджин
    申请人:Эли Лилли Энд Компани (Фирма)；
    IPC主号:
    专利说明:

    This invention relates to the microbiological industry and concerns the production of antibiotics. The aim of the invention is to obtain the antibiotic tilactone. A strain of Streptomyces fradiae NRB 12188 (Agricultural Research Cultur Collectin NBKL) is used to prepare the tilactone. The method for preparing the tylactone of formula Q. CHfCHj is that the Stretchomyces fradiae NRRL 12188 strain is grown in a nutrient medium containing a source of carbon, nitrogen and inorganic salts under aerobic fermentation conditions, followed by isolation of the desired product, and by esterifying it. Anhydrides, acid chlorides (in combination with a base or other acid-binding compound, and esters of organic acids are active) are used as acylirucine agents in the preparation of a tilactone. Acylation can also be carried out using a mixture of an organic acid and a dehydrating agent, for example, N, N -dicyclohexylcarbodiimide. Acylation can also be carried out enzymatically. The resulting acyl derivatives can be separated and purified by known methods. These derivatives can be obtained by known methods of esterification, for example, by treating the compound with a stoichiometric amount (or slight excess) of the acylating agent, for example, an organic acid anhydride in an organic solvent, for example pyridine, at 0-24 ° C for 1-24 hours until complete esterification. . The resulting esters can be isolated from the reaction mixture by known methods, for example, extraction, chromatography and crystallization. Practically significant esters include esters of organic acids, including aliphatic cycloaliphatic, aryl, aralkyl, heterocyclic carboxylic sulfonic and alkoxycarboxylic with 1-18 carbon atoms, and inorganic acids, such as sulfuric and phosphoric. The use of the OTCs is esters based on acids such as formic acid, acetyl, chloruxus, propionic, oily, izovalerianov, glukuronov, alcohols, alcohols, alcohols, etc. , phenoxy acetic, almond, 2-thienyl acetic, and alkyl, aryl, and aralkylsulfonic acids, which can be seen in the aromatic part by halo, nitro, lower alkoxy, and esters derived from ove dicarboxylic acids such as succinic, raspberry, fumaric, malonic and phthalic. Tilactone is obtained by cultivating the strain of Streptomyces fradiae. The strain Streptomyces fradiae obtained by chemical mutagenosis of the strain Streptomyces fradiae, producing tylosin. The strain is stored in the collection of strains of microorganisms of the Northern Regional Research Center Agricultural Resarch North North Region 1815 North University Strecht Peoria Illionis 61604 under registration number KRRL 12188. The strain Streptomyces fradiae grows at 10-4 ° C. As a nutrient medium for growing Streptomyces fradiae, various nutrient media are used that contain carbohydrates such as dextrin, glucose, starch, grain flour, and oils, such as soybean, as a carbon source. Grain flour, soy flour, fish meal, amine acid are used as a source of nitrogen. Soluble salts can be introduced into the nutrient medium, with the congeneric acid forming iron, potassium, sodium, magnesium, ammonium calcium, chlorine, carbonate, sulfate and nitration ions. The nutrient medium may also contain trace elements necessary for growth and development of the body. These trace elements are in the form of impurities in the composition of other components of the medium in an amount necessary for the growth of the organism. If necessary, an antifoam agent, such as polypropylene glycol with a molecular weight of about 2000, is added. To obtain significant quantities of tilactone, aerobic deep fermentation is carried out in tanks. Tilactone is prepared in small quantities in a shake grower. A vegetative inoculum is used for cultivation. A vegetative inoculum
    receive a sowing of a small amount of nutrient medium spore form of the body or mycelium to obtain a fresh, actively growing body culture. The vegetative inoculum is then transferred to a larger tank.
    For the vegetative inoculum, the same medium is used for fermentation on a large scale, but other nutrient media can be used.
    For aerobic deep cultivation, sterile air is passed. To efficiently produce an antibiotic, the percentage of air saturation should be about 30% or more at 28 ° C and 1 atm pressure.
    The obtained tilactone can be isolated from the medium by known methods (by extraction or by filtration by an enzyme broth).
    When extracting, various methods can be used. To purify the filtered broth, a method is used in which the broth is extracted without adjusting the pH to a suitable solvent, for example, amylacetate or petroleum ether, and concentrating the organic phase under vacuum to obtain crystals or oil. In oil recovery, it can be purified by adsorption chromatography.
    Example 1. A lyophilized tablet of Streptomyces fradiae is dispersed in 1-2 ml of sterilized water. A part of this solution (0.5 ml) is used to inoculate a vegetative medium (150 ml) having the following composition, wt.%: An infusion of water-soaked grain 1.0 t yeast extract 0.5-soy. whole 0.5) caso 0.3j soybean oil 0.45 and deionized water 97.25.
    Alternatively, the Streptomyces fradiae vegetative NRRL 12188, stored 1 ml in liquid nitrogen, is quickly thawed and used to inoculate the vegetative medium. The incubated vegetative medium is incubated in a 500 ml Erlenmeyer flask at 48 h in a rocking chair at a rate. rotation 300 rpm. I-.
    The obtained incubated vegetative medium in the amount of 0.5 ml is used to inoculate 7 ml of the nutrient medium with the following composition, wt.%: Beet molasses 2.0j grain flour l, 5j fish flour 0.9j gluten grain 0.9; NaC, 0.1) (Nti () jHPO Q, 04) CaCOj 0.2, soybean oil 3.0 and deionized water 91.36.
    Inoculated nutrient DU incubated in a 50 ml vessel at 6 days in a rocking chair at 300 rpm
    RNA for inoculum production in a large volume of 60 ml of an incubated vegetative medium prepared by the method described above is used to inoculate 38 liters of vegetative medium, second stage growth, having the following composition eV, wt.%: Infusion of water-soaked grain 2.0 soybean flour 0 ,five; yeast extract 0.5; CaCO 0.3 {soybean oil 0.5 lecithin 0.015 and water 97.185.
    By adding a 50% NaOH solution, the pH is adjusted to 8.5.
    The vegetative medium of the second stage is incubated in a 68 l 47 h tank at. 4 liters of the resulting incubate at 29 ° C .4 l of the second stage incubated medium are used to inoculate 40 liters of sterile medium of the following composition, wt.%: Fish meal 0.9 grain flour I, 57i gluten grain 0.92 CaCOej 0.21} NaCl 0.1 / (NHjj) HFOjij 0.04 beet molasses 2.1 | Soybean oil 3.15 - Lecithin 0.09 and water 90.9.
    The pH was adjusted to 7.2 with a 50% NaOH solution. The inoculated culture medium is fermented in a 63 L tank for 5 days at. The nutrient medium is aerated with air in such a way that the level of dissolved air is 30-50%, mixing is carried out with agitators at a speed of 300 rpm.
    Example 2. Fermented broth (1600 l), obtained by the method of example 1, is filtered through a filtering device. The pH of the filtrate was adjusted to 9 by adding 2% sodium hydroxide solution.
    The filtrate is extracted with 400 L of amylacetate. The amyl acetate extract, which has a high optical density but does not exhibit antimicrobial activity, is concentrated in vacuo to give an oil.
    The oil is dissolved in 5 L of benzene, the benzene solution is chromatographed on a column filled with silica gel and washed with benzene. Elution was monitored by thin layer chromatography, using a mixture of benzene-ethyl acetate (3: 2) as a solvent system and sprayed sulfuric acid for development. The column is first primed with benzene to remove the metal compounds, and then a mixture of benzene-ethyl acetate and (9: 1) -l to remove and isolate the tylecton. The fractions containing tilactone are combined and evaporated under vacuum. Tilekton is crystallized from a mixture of benzene-hexane or hot hexane with a yield of 2 g.
    Tilactone is a solid solid crystallized from hexane or a mixture of hexane.
    With ethyl acetate, melting at 162163 C,
    The elemental composition,%; carbon 70, hydrogen 9.7 and oxygen 20.3. The empirical formula of tilactone is CgiHjjOg, molecular weight 394.
    Infrared spectrum of tilactone in chloroform. Frequencies at which noticeable absorption maxima are detected are: 3534 (dn) 2924 (strong), 2398 (weak) 2353 (weak), 1709 (very strong), 1678 (very strong): 1626 (small), 1592 (very strong) , 1458 (strong), 1441 (shoulder), 1379 (small), 1404 (strong), 1316 (strong), 1284 (medium), 1181. (very strong), 1143 (strong), 1103 (medium), 1078 (medium), 1049 (very small), 1025 (medium), 984 (very strong), 958 (strong), 923 (medium), 911 (shoulder ), 859 (small), 868 (medium), 840 (medium), 820 (very small) and 661 cm (small).
    The absorption spectrum in the ultraviolet region in neutral ethanol has an absorption maximum at 282 nm (, 560).
     -55.23 ° (C1).
    Electrometric titration of tilactone in a 66% aqueous solution of dimethylformamide shows that there are no titrated groups in it.
    Tilactone is almost insoluble in water, but soluble in organic solvents (acetone, methanol, ethanol, dimethylformamide, chloroform, diethyl ether, benzene, and dimethyl sulfoxide).
    Tilactone can be separated with tylosin by thin layer chromatography on silica gel. For
    the manifestations used are sprayed sulfuric acid, concentrated or diluted (50%), in which case the tilactone appears first as a spot with a yellow, brownish color.
    Example 3. 3,5-Di-O-atiltiltilton.
    200 mg of tilactone, prepared according to Example 2, was dissolved in 4 ml of pyridine, 4 ml of acetic anhydride was added, the mixture was left at room temperature for 16 hours, and then evaporated to dryness in vacuo. 5 ml of methanol was added to the obtained residue, the solution was heated at 30 minutes, the nocfie of which was concentrated in vacuo to give 3,5-di-O-acetylthylactone having an IW value in thin layer chromatography with a solvent system of benzene-ethyl acetate (4: 1) on silica gel 0.59. Tilactone in the same solvent system has a Pu value of 0.3.
    Examples 4-7. Get 3,5-di-O-propionylacetate according to use 3, but using proton anhydride.
    Get 3,5-di-O-isovalerylthylactone according to example 3, but using isov; zrianic anhydride.
    The 3,5-di-O-n-butyryl tilactone is obtained according to Example 3, but using butyric anhydride.
    3,5-di-O-n-butyrylthylactone is obtained according to the method described in Example 3, but using oily anhydride.
    Therefore, the proposed method and the Streptomyces fradlae NRRL 12188 strain allow to obtain a new antibiotic tilactone.
    权利要求:
    Claims (1)
    [1]
    Get 3,5-di-0-isovalerylacetate according to example 3, but with
    30 min. Isovalic anhydride
    A 3,5-di-0-n-butyrylthylac tone is obtained according to Example 3, but using oily anhydride.
    A 3,5-di-0-n-butyrylthi35 lactone is obtained according to the method of Example 3, but using butyric anhydride.
    Therefore, the proposed method and strain igberbouzese GgasNae
    Dr. IVKB 12188 allows to get a new antibiotic tilactone.
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    引用文献:
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    法律状态:
    优先权:
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