WO2013151519A1 - Fungal strain penicillium roseopurpureum - producer of curvularin - Google Patents
Fungal strain penicillium roseopurpureum - producer of curvularin Download PDFInfo
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- WO2013151519A1 WO2013151519A1 PCT/UA2012/000033 UA2012000033W WO2013151519A1 WO 2013151519 A1 WO2013151519 A1 WO 2013151519A1 UA 2012000033 W UA2012000033 W UA 2012000033W WO 2013151519 A1 WO2013151519 A1 WO 2013151519A1
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- Prior art keywords
- curvularin
- roseopurpureum
- producer
- penicillium roseopurpureum
- penicillium
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- VDUIGYAPSXCJFC-UHFFFAOYSA-N S-curvularin Natural products C1C(=O)OC(C)CCCCCC(=O)C2=C(O)C=C(O)C=C21 VDUIGYAPSXCJFC-UHFFFAOYSA-N 0.000 title claims abstract description 24
- VDUIGYAPSXCJFC-JTQLQIEISA-N (5s)-13,15-dihydroxy-5-methyl-4-oxabicyclo[10.4.0]hexadeca-1(12),13,15-triene-3,11-dione Chemical compound C1C(=O)O[C@@H](C)CCCCCC(=O)C2=C(O)C=C(O)C=C21 VDUIGYAPSXCJFC-JTQLQIEISA-N 0.000 title claims abstract description 23
- 241000985512 Penicillium roseopurpureum Species 0.000 title claims abstract description 11
- 230000002538 fungal effect Effects 0.000 title abstract description 3
- 241000233866 Fungi Species 0.000 claims description 4
- 229930000044 secondary metabolite Natural products 0.000 abstract description 4
- 238000004519 manufacturing process Methods 0.000 abstract description 3
- 238000002360 preparation method Methods 0.000 abstract description 3
- 231100000678 Mycotoxin Toxicity 0.000 abstract description 2
- 201000010099 disease Diseases 0.000 abstract description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 abstract description 2
- 230000000855 fungicidal effect Effects 0.000 abstract description 2
- 239000000203 mixture Substances 0.000 abstract description 2
- 239000002636 mycotoxin Substances 0.000 abstract description 2
- 238000011321 prophylaxis Methods 0.000 abstract 1
- UHOVQNZJYSORNB-UHFFFAOYSA-N Benzene Chemical compound C1=CC=CC=C1 UHOVQNZJYSORNB-UHFFFAOYSA-N 0.000 description 15
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 10
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 9
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 9
- 229920001817 Agar Polymers 0.000 description 6
- 239000008272 agar Substances 0.000 description 6
- 241000124008 Mammalia Species 0.000 description 4
- 241000465255 Penicillium citreoviride Species 0.000 description 4
- 238000000605 extraction Methods 0.000 description 4
- 239000008103 glucose Substances 0.000 description 4
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 4
- 239000003053 toxin Substances 0.000 description 4
- 231100000765 toxin Toxicity 0.000 description 4
- 241000196324 Embryophyta Species 0.000 description 3
- 241000228143 Penicillium Species 0.000 description 3
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- 238000004440 column chromatography Methods 0.000 description 3
- 150000001875 compounds Chemical class 0.000 description 3
- 238000002425 crystallisation Methods 0.000 description 3
- 230000008025 crystallization Effects 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 238000001704 evaporation Methods 0.000 description 3
- 230000008020 evaporation Effects 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 230000002588 toxic effect Effects 0.000 description 3
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 2
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 2
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 2
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 2
- 229910052799 carbon Inorganic materials 0.000 description 2
- 239000000284 extract Substances 0.000 description 2
- 239000000706 filtrate Substances 0.000 description 2
- 238000002955 isolation Methods 0.000 description 2
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 239000000741 silica gel Substances 0.000 description 2
- 229910002027 silica gel Inorganic materials 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- 231100000331 toxic Toxicity 0.000 description 2
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 description 1
- 235000001674 Agaricus brunnescens Nutrition 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 235000007516 Chrysanthemum Nutrition 0.000 description 1
- 240000005250 Chrysanthemum indicum Species 0.000 description 1
- JLSVDPQAIKFBTO-OMCRQDLASA-N Citreoviridin Chemical compound COC1=CC(=O)OC(\C=C\C=C\C=C\C(\C)=C\[C@@]2(C)[C@@H]([C@@](C)(O)[C@@H](C)O2)O)=C1C JLSVDPQAIKFBTO-OMCRQDLASA-N 0.000 description 1
- 240000007049 Juglans regia Species 0.000 description 1
- 235000009496 Juglans regia Nutrition 0.000 description 1
- WEVYAHXRMPXWCK-UHFFFAOYSA-N acetonitrile Substances CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 1
- LMDSAFNXCHPEQY-UHFFFAOYSA-N acetonitrile hexane Chemical compound CCCCCC.C(C)#N.C(C)#N LMDSAFNXCHPEQY-UHFFFAOYSA-N 0.000 description 1
- 238000012271 agricultural production Methods 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- BXUGQFINZUXAKN-UHFFFAOYSA-N citreoviridin Natural products COC1=CC(=O)OC(=C1C)C=CC=CC=CC=CC2(C)OC(C)C(C)(O)C2O BXUGQFINZUXAKN-UHFFFAOYSA-N 0.000 description 1
- JLSVDPQAIKFBTO-UHFFFAOYSA-N citreoviridin A Natural products COC1=CC(=O)OC(C=CC=CC=CC(C)=CC2(C)C(C(C)(O)C(C)O2)O)=C1C JLSVDPQAIKFBTO-UHFFFAOYSA-N 0.000 description 1
- 239000003086 colorant Substances 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 239000003480 eluent Substances 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 235000019253 formic acid Nutrition 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- 244000005706 microflora Species 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 230000003032 phytopathogenic effect Effects 0.000 description 1
- 239000004476 plant protection product Substances 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 238000004062 sedimentation Methods 0.000 description 1
- 238000010187 selection method Methods 0.000 description 1
- 238000010898 silica gel chromatography Methods 0.000 description 1
- 239000002594 sorbent Substances 0.000 description 1
- 230000003595 spectral effect Effects 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 231100000563 toxic property Toxicity 0.000 description 1
- 235000020234 walnut Nutrition 0.000 description 1
Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
- C12N1/145—Fungal isolates
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
- C12P17/02—Oxygen as only ring hetero atoms
- C12P17/08—Oxygen as only ring hetero atoms containing a hetero ring of at least seven ring members, e.g. zearalenone, macrolide aglycons
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/645—Fungi ; Processes using fungi
- C12R2001/80—Penicillium
Definitions
- the invention relates to the field of microbiology and relates to the production of a new strain of the fungus Penicillium roseopurpureum, which can be used to produce mycotoxin (secondary metabolite) curvularin, which is used as a part of preparations with fungicidal activity for the prevention and protection against diseases of medicinal plants, garden, ornamental and other agricultural cultures.
- Penicillium citreo-viride S. Kosemura - Meroterpenoids From Penicillium citreo-viride B. IFO 4692 and 6200 Hybrid / / Tetrahedron, 2003, v. 59, W 27, pp. 5055-5072
- curvularin a secondary metabolite citreoviridin
- the basis of the invention is the task to obtain a strain of the fungus Penicillium roseopurpureum - producer of curvularin, using selective selection methods from the natural population of the strain, which, having the ability to synthesize curvularin and its hydroxycurvularin derivative, is characterized by the fact that it does not produce other metabolites other than curvularin allows using it to obtain preparations suitable for environmentally friendly plant protection technologies.
- Penicillium roseopurpureum the producer of curvularin, which is isolated from the epiphytic microflora of chrysanthemums, the fungus was identified as Penicillium roseopurpureum (Dierckx, 1901), the Monoverticillata lanata section, a standard method of mycological research, deposited in microbiology and deposited in D.K. Zabolotnogo NAS of Ukraine with registration number Penicillium roseopurpureum 1MB F-100055 (March 22, 2010).
- the proposed (deposited) strain acquires such cultural-morphological features and physiological and biochemical properties that ensure the production of curvularin, which does not exhibit toxic properties with respect to to mammals and, as part of plant protection products, is environmentally friendly.
- the resulting strain has the following cultural and morphological characteristics and physiological and biochemical properties.
- Conidiophores short 50 x 1.5 - 2 ⁇ , depart from branching of intertwined air hyphae, sometimes from hyphae submerged in agar, then they reach up to 100 ⁇ long, rarely branched, smooth-walled, at the apex with monoverticulate brushes, which consist of 8 - 20 sterigm, 6-7 x 1,5-2 ⁇ .
- Conidia are spherical, slightly rough, 2–2.5 ⁇ , quickly fall away from the sterigm. Reversum orange-red, hot pink or brick-colored agar painted in the same colors, but less intense.
- the optimum growth temperature of the strain is + (25-28) 0 C, and grows at higher temperatures up to +32 ° C and at low temperatures up to +5 ° C.
- the optimum temperature of toxin formation is + 26 ° C.
- test cultures including phytopathogenic organisms
- KGA potato-glucose agar
- the resulting strain absorbs glucose well.
- Example ⁇ ° 1 The fungus Penicillium roseopurpureum, the producer of curvularin, was cultured in 700 ml Erlenmeyer flasks containing 100 ml of Capek liquid medium with glucose (20 g / l) as the only carbon source, for 21 days at a temperature of (27-28) 0 ⁇ without access to light. From the obtained culture filtrates of this fungus by triple extraction with chloroform and subsequent evaporation of the extracts, an oily residue was obtained, after preliminary purification from non-polar lipid impurities using liquid-liquid redistribution (n-hescan-acetonitrile), was fractionated using column chromatography.
- Curvularin content was determined by extraction and column chromatography of the active compounds / extractants: chloroform or ethyl acetate; dynamics chromatography: sorbent silica gel of the II degree of activity according to Brockman (100/160 microns) eluent: n-hexane, chloroform, acetone and their systems; crystallization: from benzene or a mixture of methanol: n-hexane. With one kilogram of the substrate after the above toxin formation, curvularin is obtained in an amount of 16 g / l.
- the studied physicochemical and spectral characteristics of the isolated compounds provide grounds for their identification with curvularin and its hydroxycurvularin derivative.
- Example N Curvularin content was determined by the crystalline yield of the drug. After biosynthesis of a producer fungus on a grain substrate, the isolation of curvularin from the native culture was carried out by triple extraction with chloroform, followed by evaporation. Isolation of the toxin from the extract included purification by liquid-liquid redistribution in the acetonitrilehexane system followed by re-extraction of the toxin in the benzene: ethyl acetate: methanol (5: 5: 1) and toluene: isopropanol: formic acid (6: 3: 1) systems, drying, evaporation sedimentation to a minimum volume of silica gel and column chromatography.
- the content of curvularin-containing fractions was combined and mitotoxin was crystallized at room temperature.
- the yield of curvularin by crystallization from benzene is 82.6 mg, and hydroxycurvularin 12.7 mg with 5 l of culture fungal filtrate.
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- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Heterocyclic Carbon Compounds Containing A Hetero Ring Having Oxygen Or Sulfur (AREA)
Abstract
The invention relates to the field of microbiology and concerns the production of a novel fungal strain Penicillium roseopurpureum which can be used for producing the mycotoxin (secondary metabolite) Curvularin, which is used in the composition of preparations having fungicidal activity for the prophylaxis and protection of medicinal plants and horticultural, decorative and other agricultural crops from diseases. The strain Penicillium roseopurpureum - a producer of Curvularin, has been deposited in the depository at the D. K. Zabolotny Institute of Microbiology and Virology of the National Academy of Sciences of Ukraine under registration number Penicillium roseopurpureum IMB F- 100055 (22 March 2010).
Description
ШТАММ ГРИБА PENICILLIUM STRAIN MUSHROOM PENICILLIUM
ROSEOPURPUREUM - ПРОДУЦЕНТ КУРВУЛАРИНА ROSEOPURPUREUM - PRODUCER OF CURVULARINE
Изобретение относится к области микробиологии и касается получения нового штамма гриба Penicillium roseopurpureum, который может быть использован для получения микотоксина (вторичного метаболита) курвуларина, что в составе препаратов с фунгицидной активностью применяется для профилактики и защиты от болезней лекарственных растений, садовых, декоративных и других сельскохозяйственных культур. The invention relates to the field of microbiology and relates to the production of a new strain of the fungus Penicillium roseopurpureum, which can be used to produce mycotoxin (secondary metabolite) curvularin, which is used as a part of preparations with fungicidal activity for the prevention and protection against diseases of medicinal plants, garden, ornamental and other agricultural cultures.
Известны штаммы Penicillium citreo-viride, Penicillium baradicum и ряд других видов штаммов - продуцентов курвуларина. (Vesonder RF, Ciegler A., Fennell D., Tjarks LW, Jensen AH - Curvularin from Penicillium baradicum baghdadi NRRL 3754, and biological effects. / / Journal of environmental science and health. J Environ Sci Health Part В Pestic Food Contam Agric Wastes 1976, 11 (4) p. 289-297; S. Kosemura - Meroterpenoids From Penicillium citreo-viride B. IFO 4692 and 6200 Hybrid / / Tetrahedron, 2003, v.
Known strains of Penicillium citreo-viride, Penicillium baradicum and several other types of strains - producers of curvularin. (Vesonder RF, Ciegler A., Fennell D., Tjarks LW, Jensen AH - Curvularin from Penicillium baradicum baghdadi NRRL 3754, and biological effects. / / Journal of environmental science and health. J Environ Sci Health Part In Pestic Food Contam Agric Wastes 1976, 11 (4) p. 289-297; S. Kosemura - Meroterpenoids From Penicillium citreo-viride B. IFO 4692 and 6200 Hybrid / / Tetrahedron, 2003, v.
Известные штаммы имеют меньшую способность к синтезу курвуларина и его производного гидрооксикурвуларина, а также характеризуются тем, что производят, кроме курвуларина, другие вторичные метаболиты, которые являются токсичными для млекопитающих, что ограничивает возможность их использования для защиты растений. В настоящее время в сельскохозяйственной микробиологии неизвестны штаммы-продуценты курвуларина, имеющие
более высокую активность в готовом препарате и не токсичные для млекопитающих. Known strains have less ability to synthesize curvularin and its hydroxycurvularin derivative, and are also characterized by the fact that they produce, in addition to curvularin, other secondary metabolites that are toxic to mammals, which limits the possibility of their use for plant protection. Currently, in agricultural microbiology, the curvularin producing strains having higher activity in the finished product and non-toxic to mammals.
Наиболее близким к предложенному штамму является Penicillium citreo-viride (S. Kosemura - Meroterpenoids From Penicillium citreo-viride B. IFO 4692 and 6200 Hybrid / / Tetrahedron, 2003, v. 59, Ш 27, pp. 5055-5072), который, кроме курвуларина, производит вторичный метаболит цитреовиридин, он токсичен для млекопитающих, что препятствует его использованию в сельскохозяйственном производстве. Closest to the proposed strain is Penicillium citreo-viride (S. Kosemura - Meroterpenoids From Penicillium citreo-viride B. IFO 4692 and 6200 Hybrid / / Tetrahedron, 2003, v. 59, W 27, pp. 5055-5072), which in addition to curvularin, it produces a secondary metabolite citreoviridin, it is toxic to mammals, which prevents its use in agricultural production.
В основу изобретения поставлена задача получить штамм гриба Penicillium roseopurpureum - продуцент курвуларина, путем использования методов селективного отбора из природной популяции штамма, который, имея способность к синтезу курвуларина и его производного гидрооксикурвуларина, характеризуется тем, что не производит, кроме курвуларина, другие метаболиты, это позволяет при его использовании получать препараты пригодные для экологически чистых технологий защиты растений. The basis of the invention is the task to obtain a strain of the fungus Penicillium roseopurpureum - producer of curvularin, using selective selection methods from the natural population of the strain, which, having the ability to synthesize curvularin and its hydroxycurvularin derivative, is characterized by the fact that it does not produce other metabolites other than curvularin allows using it to obtain preparations suitable for environmentally friendly plant protection technologies.
Поставленная задача решается путем получения нового штамма гриба Penicillium roseopurpureum - продуцента курвуларина, который выделен из эпифитной микрофлоры хризантем, гриб был идентифицирован как Penicillium roseopurpureum (Dierckx, 1901), секция Monoverticillata lanata, стандартным методом микологических исследований, депонированный в депозитарии Института микробиологии и вирусологии имени Д.К.Заболотного НАН Украины с регистрационным номером Penicillium roseopurpureum 1MB F- 100055 (22 марта 2010 года). The problem is solved by obtaining a new strain of the fungus Penicillium roseopurpureum - the producer of curvularin, which is isolated from the epiphytic microflora of chrysanthemums, the fungus was identified as Penicillium roseopurpureum (Dierckx, 1901), the Monoverticillata lanata section, a standard method of mycological research, deposited in microbiology and deposited in D.K. Zabolotnogo NAS of Ukraine with registration number Penicillium roseopurpureum 1MB F-100055 (March 22, 2010).
В результате серии отборов предложеный (депонированный) штамм приобретает такие культурально-морфологические признаки и физиолого- биохимические свойства, обеспечивающие выработку курвуларина, который не проявляет токсические свойства по отношению к
млекопитающим и, в составе препаратов защиты растений, является безопасным к окружающей среде. As a result of a series of selections, the proposed (deposited) strain acquires such cultural-morphological features and physiological and biochemical properties that ensure the production of curvularin, which does not exhibit toxic properties with respect to to mammals and, as part of plant protection products, is environmentally friendly.
Полученный штамм имеет следующие культурально- морфологические признаки и физиолого-биохимические свойства. The resulting strain has the following cultural and morphological characteristics and physiological and biochemical properties.
Культивируется на средах Чапека, Ваксмана, картофельно-глюкозном агаре (КГ А), сусло-агаре. Оптимальная температура культивирования - 25 - 28 0 С. Максимальная скорость роста наблюдается в КГА (0,26 мм / час), меньше на сусло-агаре (0,21 мм / час), наименьшая скорость роста на среде Ваксмана (0,19 мм / час). На среде Чапека колонии растут медленнее (скорость роста составляет 0,18 мм / час). Колонии в центральной части повышенные, бороздчатые, серовато-белые, бледно-розовые, серовато- зеленые до орехово-зеленых. Конидиеносцы короткие 50 х 1,5 - 2 μ, отходят от ветвления переплетенных между собой воздушных гиф, иногда от погруженных в агар гиф, тогда они достигают до 100μ длиной, редко разветвленные, гладкостенные, на верхушке с моновертицилятными кисточками, которые состоят из 8 -20 стеригм, 6-7 х 1,5-2μ. Конидии шаровидные слабошероховатые 2-2,5 μ, быстро отпадают от стеригм. Реверзум оранжево-красный, ярко розовый или кирпичного цвета агар окрашен в те же цвета, но менее интенсивно. It is cultivated on Chapek, Waxman, potato-glucose agar (KG A), wort-agar. The optimum cultivation temperature is 25 - 28 0 С. The maximum growth rate is observed in the KGA (0.26 mm / h), less on wort agar (0.21 mm / h), the lowest growth rate on Waxman’s medium (0.19 mm / hour). On Chapek’s environment, colonies grow more slowly (growth rate is 0.18 mm / h). The colonies in the central part are elevated, furrowed, grayish-white, pale pink, grayish green to walnut green. Conidiophores short 50 x 1.5 - 2 μ, depart from branching of intertwined air hyphae, sometimes from hyphae submerged in agar, then they reach up to 100 μ long, rarely branched, smooth-walled, at the apex with monoverticulate brushes, which consist of 8 - 20 sterigm, 6-7 x 1,5-2μ. Conidia are spherical, slightly rough, 2–2.5 μ, quickly fall away from the sterigm. Reversum orange-red, hot pink or brick-colored agar painted in the same colors, but less intense.
Оптимальная температура роста штамма + (25-28) 0 С, растет и при более высоких температурах до +32 ° С и при низких температурах до +5 ° С. Оптимальная температура токсинообразования + 26 ° С. The optimum growth temperature of the strain is + (25-28) 0 C, and grows at higher temperatures up to +32 ° C and at low temperatures up to +5 ° C. The optimum temperature of toxin formation is + 26 ° C.
Наибольшая активность штамма к тест культурам (в том числе и фитопатогенных организмов) наблюдается на 21 сутки культивирования на КГА (картофельно-глюкозного агаре). The highest strain activity for test cultures (including phytopathogenic organisms) is observed on day 21 of cultivation on KGA (potato-glucose agar).
Полученный штамм хорошо усваивает глюкозу. The resulting strain absorbs glucose well.
Пример Ν° 1.
Гриб Penicillium roseopurpureum-продуцент курвуларина культивировали в колбах Эрленмейера объемом 700 мл, содержащих 100 мл жидкой среды Чапека с глюкозой (20 г / л) как единственным источником углерода, в течение 21 суток при температуре (27-28) 0 С без доступа света. Из полученных культуральных фильтратов этого гриба путем трехкратной экстракции хлороформом и последующим упариванием экстрактов, был получен маслянистый остаток, после предварительной очистки от неполярных липидных примесей с помощью жидкость-жидкостного перераспределения (н-гескан-ацетонитрил), фракционировали с применением колоночной хроматографии. Элюция из колонки показала, что фракции No JNb 7-10 (хлороформ) имеют высокую активность в отношении индикаторных культур, а хроматографически представлены одним пятном с Rf 0,56 (система бензол: этилацетат: метанол, 5: 5: 1). Эти фракции объединяли и упаривали под пониженным давлением. Путем кристаллизации из бензола было получено 82,6 мг соединения белого цвета, которое было обозначено как Рг 10. Example Ν ° 1. The fungus Penicillium roseopurpureum, the producer of curvularin, was cultured in 700 ml Erlenmeyer flasks containing 100 ml of Capek liquid medium with glucose (20 g / l) as the only carbon source, for 21 days at a temperature of (27-28) 0 С without access to light. From the obtained culture filtrates of this fungus by triple extraction with chloroform and subsequent evaporation of the extracts, an oily residue was obtained, after preliminary purification from non-polar lipid impurities using liquid-liquid redistribution (n-hescan-acetonitrile), was fractionated using column chromatography. Elution from the column showed that fractions No JNb 7-10 (chloroform) are highly active against indicator cultures and are chromatographically represented by one spot with Rf 0.56 (benzene: ethyl acetate: methanol system, 5: 5: 1). These fractions were combined and evaporated under reduced pressure. 82.6 mg of a white compound, which was designated as Pr 10, was obtained by crystallization from benzene.
Содержание курвуларина определяли методом экстракции и колоночной хроматографии активных соединений / экстрагентов: хлороформ или этилацетат; динамиком хроматография: сорбент силикагель II степени активности по Брокману (100/160 мкм) элюента: н-гексан, хлороформ, ацетон и их системы; кристаллизации: из бензола или смеси метанол: н- гексан. С одного килограмма субстрата после вышеперечисленного токсинообразования получается курвуларин в количестве 16 г \ л. Curvularin content was determined by extraction and column chromatography of the active compounds / extractants: chloroform or ethyl acetate; dynamics chromatography: sorbent silica gel of the II degree of activity according to Brockman (100/160 microns) eluent: n-hexane, chloroform, acetone and their systems; crystallization: from benzene or a mixture of methanol: n-hexane. With one kilogram of the substrate after the above toxin formation, curvularin is obtained in an amount of 16 g / l.
Исследованые физико-химические и спектральные характеристики выделенных соединений (t 0 С, UV-и РЖ-спектры) дают основания для идентификации их с курвуларином и его производным гидрооксикурвуларином. The studied physicochemical and spectral characteristics of the isolated compounds (t 0 C, UV and RG spectra) provide grounds for their identification with curvularin and its hydroxycurvularin derivative.
Пример N° 2.
Содержание курвуларина определяли по кристаллическому выходу препарата. После биосинтеза гриба - продуцента на зерновом субстрате, выделение курвуларина с нативной культуры проводили путем трехкратной экстракции хлороформом с последующим упариванием. Выделение токсина из экстракта включало очистку путем жидкость- жидкостного перераспределения в системе ацетонитрилгексан с последующей переэкстракцией токсина в системе бензол: этилацетат: метанол (5:5: 1) и толуол: изопропанол: муравьиная кислота (6:3: 1), высушиванию, упариванию, осаждению до минимального объема селикагеля и хроматографией на колонке. Содержание курвуларинсодержащих фракций объединяли и проводили кристаллизацию митотоксина при комнатной температуре. Выход курвуларина путем кристаллизации из бензола составляет 82.6 мг, а гидрооксикурвуларина 12,7 мг с 5 л культурального фильтрата гриба.
Example N ° 2. Curvularin content was determined by the crystalline yield of the drug. After biosynthesis of a producer fungus on a grain substrate, the isolation of curvularin from the native culture was carried out by triple extraction with chloroform, followed by evaporation. Isolation of the toxin from the extract included purification by liquid-liquid redistribution in the acetonitrilehexane system followed by re-extraction of the toxin in the benzene: ethyl acetate: methanol (5: 5: 1) and toluene: isopropanol: formic acid (6: 3: 1) systems, drying, evaporation sedimentation to a minimum volume of silica gel and column chromatography. The content of curvularin-containing fractions was combined and mitotoxin was crystallized at room temperature. The yield of curvularin by crystallization from benzene is 82.6 mg, and hydroxycurvularin 12.7 mg with 5 l of culture fungal filtrate.
Claims
Формула изобретения. Штам гриба PeniciUimn roseopurpureum - продуцент курвуларина депонированный в депозитарии Института микробиологии и вирусологии имени Д.К.Заболотного НАН Украины с регистрационным номером Penicillium roseopurpureum 1MB F- 100055 (22 марта 2010 года).
Claim. The fungus strain PeniciUimn roseopurpureum is a producer of curvularin deposited with the depository of the D.K. Zabolotny Institute of Microbiology and NAS of Ukraine with the registration number Penicillium roseopurpureum 1MB F-100055 (March 22, 2010).
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| CN115044483A (en) * | 2022-05-23 | 2022-09-13 | 浙江工业大学 | Penicillium W21C371 and its application in the preparation of curvilinear |
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Non-Patent Citations (3)
| Title |
|---|
| DATABASE MEDLINE accession no. 002951 * |
| SHENG LAI ET AL.: "Novel curvularin-type metabolites of a hybrid strain ME 0005 derived from Penicillium citreo-viride B. IFO 6200 and 4692", TETRAHEDRON LETTERS, vol. 30, no. 17., 1989, pages 2241 - 2244, XP055081997, Retrieved from the Internet <URL:http://www.sciencedirect.com/science/article/pii/S0040403900996588> [retrieved on 20120618], doi:10.1016/S0040-4039(00)99658-8 * |
| VESONDER R.F. ET AL.: "Curvularin from penicillium baradicum Baghdadi NRRL 3754, and biological effects", J ENVIRON SCI HEALTH B., vol. 1, no. 4, 1976, pages 289 - 297, XP055082003, doi:10.1080/03601237609372044 * |
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| CN115044483A (en) * | 2022-05-23 | 2022-09-13 | 浙江工业大学 | Penicillium W21C371 and its application in the preparation of curvilinear |
| CN115044483B (en) * | 2022-05-23 | 2023-09-05 | 浙江工业大学 | Penicillium fungus W21C371 and application thereof in preparation of curvulin |
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