CS245153B1 - Microorganism strain Acremonium chrysogenum - Google Patents
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Abstract
Produkčný kmeň mikroorganizmu Acremonium chrysogenura/Thirum et Sukap/ W. Gams CCM F-729 produkuje za aerobných podmienok kyselinu 7-/D-5-amino-5-karboxy-n-valeramido/-3-acetoxymetyl-3-cefem-4-karboxylovů tzv. cefalosporín C vzorca I HOOC-CH -/ch2/3-CO-NH nh2 COOH ch2ococh3 /1/ Cefalosporín C sa používá na přípravu polosyntetickych cefalosporínových antibiotik. Cefalosporín C sa připravuje submerznou fermentáciou v přítomnosti zdrojov síry. Vdčšina priemyselných produkčných kmeftov sd auxotrofné mutanty dependentné na DL-metionín. Kmeň Acremonium chrysogenum CCM F-729 nie je závislý na DL-metioníne, produkuje cefalosporín C na ekonomicky výhodných zdrojoch síry, akým je NajS^O .51^0.The production strain of the microorganism Acremonium chrysogenum/Thirum et Sukap/ W. Gams CCM F-729 produces under aerobic conditions 7-/D-5-amino-5-carboxy-n-valeramido/-3-acetoxymethyl-3-cephem-4-carboxylic acid, so-called cephalosporin C of the formula I HOOC-CH -/ch2/3-CO-NH nh2 COOH ch2ococh3 /1/ Cephalosporin C is used for the preparation of semi-synthetic cephalosporin antibiotics. Cephalosporin C is prepared by submerged fermentation in the presence of sulfur sources. Most industrial production strains are auxotrophic mutants dependent on DL-methionine. The strain Acremonium chrysogenum CCM F-729 is not dependent on DL-methionine, it produces cephalosporin C on economically advantageous sulfur sources, such as NajS^O .51^0.
Description
245153 2
Vynález sa týká nového priemyselného mikroorganizmu produkujúceho kyselinu 7-/D-5-amino--5-karboxy-n-valeramido/-3-acetoxymetyl-3-cefem-4-karboxylovú, obecného vzorca I, tzv.cefalosporín C /CFS-C/
HOOC-CH-ICHjh-CO-NH I ' nh2 ( ch2ococh3 /1/
COOH
Cefalosporín C sa používá ako východzia surovina pri príprave polosyntetických cefalos-porínovích antibiotik. Produkčný kmeft bol získaný v screeningovom programe, zameranom naizoláciu kmeňov nezávislých na DL-metioníne. Východzím kmeftom bola kultúra Cephalosporium acremonium WIS OP 163, maximálna produkciacefalosporinu C dosahovaná na pódach s tiosíranom sodným ako zdrojom síry bola 200-400 /ig/ml.Cefalosporínové antibiotiká odvodené od kyseliny 7-aminocefalosporánovej sú vyráběné prevážnepomocou kmeňa Cephalosporium acremonium /po reklasifikácii Gamsom W. v roku 1971 Acremoniumchrysogenum/.
Rod Cephalosporium je v systematike zaradený do čelade Moniliceae do triedy Fungi imper-fecti /Deuteromycetes/. U rodu Cephalosporium sú spory vytvárané len terminálne na nevětve-ných konídiofóroch, skoro pravidelné posadených na hlavnom mycéliu. Cephalosporium tvoříseptované hýfy, septy delia protoplazmu na menšie úseky.
Konídionosiče sú jednotlivé, silné bazofilné. Známa je reprodukcia nepohlavnou mitózoua buněčnou elongáciou. Nepohlavné spory sú jednobuněčné konédie s jedným jadrom, vytváranéna hýfach.Buňky mycélia aj konídie obsahujú po jednom haploidnom jadre. Tieto vlastnostisi ponechávájú aj pri submerzných kultiváciách.
Cephalosporium ako eukariotický organizmus má membránou ohraničené jádro s chromozomálnouštruktúrou a cytoplazmou s poměrně komplexmou membránovou štruktúrou /mitochondrie, endo-plazmatické retikulum/. Významná je najma mimobunečná akumulácia antibiotika spósobená ex-kréciou cez koncentračný gradient. V submerznej kultúre sú přítomné štyri typy buníek: septované hýfy, konídie, klíčiacekonídie a artrospóry.
Morfológia je rozdielna v závislosti od použitého zdroja síry. Pri fermentácii s použi-tím DL-metionínu je převaha artrospór, s existenciou ktorých je spojená maximálna tvorbaantibiotika. Pri použití anorganických zdrojov síry sú v submerznej kultúre v prevahe vlák-nité formy; hrtíbka vlákna je menšia ako pri použití DL-metionínu.
Popisovaný kmeft bol získaný nasledujúcim spósobomi Póvodný kmeft Caphalosporium acremonium WIS OP-163 bol spracovaný chemickým mutagénomN-nitrozometylbiuretom. Najlepší izolát pracovně označený 8c VULM produkoval v submerznejkultivácii 1 800 až 2 000 .ug/ml cefalospor inu C.
Izolát 8c VULM bol v ďalšej fáze vystavený posobeníu 0V žiarenia. Získané izolátyboli hodnotené rýchlou screeningovou metodou na valčekoch z agarizovanej živnej pódys róznymi zdrojmi síry. Valčeky s vyrastenými kolonami na povrchu boli testované na agarovejplatní, s testovacím mikroorganizmom Alcaligenes faecalis ATCC 8750, Specifickým pre cefa-lospor ín C.
Izolát s nSjvSčším priemerom inhibičnej zóny na agarizovanej pode s tiosíranom sodným bolďalej testovaný v baničkovej fermentácii a v laboratórnych fermentoroch. Izolát mal pracovnéoznačenie 188 VULM, aktivita cefalosporinu C vo filtrátoch fermentačnej pódy bola stanovená 245153 v 168. kultivačnej hodině spektrofotometrickou metodou /Claridge C. A., Vaughan R. W.:
Antimicrob. Agents Chemother., 1969, 131/ a metodou HPLC /vypracovaná VÚLM/. Genealogická schéma kmeňa je označená ako schéma č. 1. Xzolát 188 VULM je uložený v Cs. sbírce mikro- organizmu UJEP v Brně pod číslom CCM F-729.
Posóbenie chemického mutagénu.
Kmeft OP 163 WIS bol deponovaný vo formě lyofilyzovanej konzervy alebo krátkodobé našikmom agare /sporulačná póda označená NB-II; zloženie uvedené v dalšom texte. Zo šikméhoagaru bola připravená vodná suspenzia o koncentrácii 10$ až 10® konídií/ml. Suspenzia bola ' přefiltrovaná cez vrstvu vaty. Mutagén N-nitrozometylbiuret bol přidaný v koncentrácii0,4 i k suspenzi! konídií.
Mutangén bol v kontakte s kultúrou počas jednej hodiny na trepacom stroji /4Hz/ pri tep-lotě 27 °C. Po uplynutí expozície bolí zo zmesi mutangén-kultúra připravené riedenia 10"?^ až10 konídií/ml. Nariedenými suspenziami boli očkované Petriho misky s agarovou pódou NB-II.Ako kontrola slúžila suspenzia spor bez pridania mutangénu.
Operácie s mutangénom prebiehali bez přístupu denného světla. Spoluprácia na Petrihomiskách trvala pri teplote 27 °C 11 dní. Jednotlivé kolonie boli přenesené na šikmý agarNB-II a odskúšané na produkčnú aktivitu na baničkách. Pósobenie fyzikálneho mutangénu: Z kultúry na šikmom agare bola připravená suspenzia konídií zmytím povrchu 10 ml steril-ně j vody. Suspenzia bola přefiltrovaná cez vatový filter; zo základnéj suspenzie boli prip- -12 -13 ravené pracovně riedenia 10 až 10 konídií/ml, ktorými boli naočkované Petriho misky/póda NB-II/. Zdrojom žiarenia bola germicídna OV-lampa /Philips TUV 30W, vlnová dlžka253,7 nm sa podiela na celkovej spektrálnej energii 93,5 %/.
Povrch agaru s nanesenými konídiami bol exponovaný počas 1 minuty zo vzdialenosti 30 cm.Po ožiarení boli Petriho misky umiestnené v termostate, kde kultúra sporulovala 11 dní priteplote 27 °C. Z vyrastených kolonií boli ihlovou technikou přenesené spory na povrch aga-rových valčekov-bločkov.
Po 5 dftoch bol povrch bločkov pokrytý kultúrou. Bločky boli umiestnené na skleněnéplatné s agarovou pódou s obsahom 25 % kultúry Alcaligenes faecalis ATCC 8750. Inkubáciabločkov trvala 18 hodin pri teplote 37 °C. Hodnotenie izolátov bolo založené na meraníinhibičných zón v okolí bločkov. Najlepší izolát bol z póvodnej kolonie na Petriho miskevyočkovaný na šikmý agar NB-II a odskúšaný v laboratórnej aj štvrťprevádzkovej fermentácii.
Agarová póda NB-II hmot./obj. Basto-beef extrakt 0,3 Bacto-pepton 0,5 glukóza 1,0 FeSO4.7H2O 0,005 MgSO4-7H2O 0,005 DL-metionín 0,005 L-cystein 0,005 agar Difco 2,5 pH pódy před sterilizáciou -6,8.
245153 4 Příprava vegetativneho inokula:
Používali sa 500 ml banky plněné 60 ml inokulačnej pódy o zložení: sacharóza 1,5 % hmot./obj. kukuřičný výluh 0,5 octan amonný 0,5 sojový olej 0,2 pH před. sterilizáciou sa upravuje na 7,0.
Na očkovanie sa používá kultúra zo šikmého agaru alebo z jačmenných zrn, tzv. jačmennékonzervy. Příprava vegetativneho inokula trvá 72 hodin pri teplote 27 °C na trepaoom stroji/4 Hz/. Vegetatívnym inokulom sa očkujú produkčně pódy rozplnené v 500 ml bankách po 40 mlo zložení: kukuřičný škrobarašídová múkasacharózaglukózaCaCO3
Na2S2°3-5H2° borax kašalotový olej 5,0 % hmot./obj7,00,51,250,50,90,052,8
Na očkovanie sa používá 8 % obj./obj. vegetativneho inokula. Kultivácia má podmienkytotožné s přípravou vegetativneho inokula, dlžka kultivácie 168 hodin. Porovnanie produkčnejaktivity kmeňa Cephalosporium acremonium WIS OP-163 s izolátmi 8c VULM a 188 VULM je v ta-bulke 1. Porovnané sú výsledky dosiahnuté v baničkovej fermentácii:
Tabulka 1 číslo experimentu kmeň prod. aktivita/yg/ml/ CFS-C 1 WIS OP-163 350 8c VULM 1 860 2 8c VULM 1 930 188 VULM 4 000 3 188 VULM 4 000 9 VULM 1 680
Kmen používaný vo fermentácii s použitím DL-metionínu ako zdroja síry. V laboratórnych skleněných tančíkoch /Výv. dielne ČSAV/, objem 2 litre, pracovný objem1 liter, bola pri podmienkach: vzdušnenie 0,75 objemu vzduchu/objem pódy, miešanie 10 Hz,teplota do 24 kultivačnej hodiny 28 °C, potom do konca fermentácie 23 °C, dosiahnutá pro-dukčně aktivita 5 200 ug/ml za 168 hodin kultivácie.
Morfológia kmeňa:
Morfológiu kmeňa Acremonium chysogenum CCM F-729 je možné popísať nasledujúcim spósobom:Pri monokolÓniovom rozseve tvoří krémové v střede béžovo sfarbené kolonie o priemere 5 až7 mm. Povrch kolonie je sline vrásčitý, okraje radiálně zbrázděné, střed kolonií je vyvýšený
245153 2
The present invention relates to a novel industrial microorganism producing 7- (D-5-amino-5-carboxy-n-valeramido) -3-acetoxymethyl-3-cephem-4-carboxylic acid of general formula (I), called cephalosporin C / CFS- C/
HOOC-CH-CHCH-CO-NH I 'nh2 (ch2ococh3 / 1 /
COOH
Cephalosporin C is used as a starting material in the preparation of semi-synthetic cephalosporin antibiotics. The production plot was obtained in a screening program aimed at isolating DL-methionine-independent strains. The starting strain was the Cephalosporium acremonium WIS OP 163 culture, the maximum production of phalosporin C produced on the sodium thiosulfate as a sulfur source was 200-400 µg / ml. in 1971 Acremoniumchrysogenum /.
The genus Cephalosporium is classified as Fungi imperfecti / Deuteromycetes in the Moniliceae family. In the genus Cephalosporium, the spores are formed only terminally on the non-branched conidiorophages, almost regular on the main mycelium. Cephalosporium formed by secreted hippies, septa divides protoplasm into smaller sections.
Conidionic agents are single, strong basophilic. Reproduction of asexual mitosis and cell elongation is known. Asexual spores are single-celled, unicellular cones, formed by the bull. Both mycelium and conid cells contain one haploid nucleus. They also retain these properties in submerged cultures.
Cephalosporium as a eukariotic organism has a membrane-bound nucleus with chromosomal structure and cytoplasm with a relatively complex membrane structure / mitochondria, endo-plasma reticulum /. Of particular note is the extracellular accumulation of the antibiotic due to excretion through the concentration gradient. Four types of cells are present in the submerged culture: septic hyphae, conidia, keyaceconidia and arthospores.
Morphology is different depending on the sulfur source used. In the DL-methionine fermentation, the predominance of arthospore is predominant, with the maximum formation of antibiotic associated with it. In the use of inorganic sulfur sources, fiber form is predominant in the submerged culture; the fiber throat is smaller than that of DL-methionine.
The described process was obtained by the following method: The original Caphalosporium acremonium WIS OP-163 was treated with the chemical mutagen N-nitrosomethylbiuret. The best isolate labeled 8c VULM produced in submerged cultures 1,800 to 2,000 µg / ml cephalosporin C.
Vulm 8c isolate was exposed to 0V radiation in the next phase. The isolates obtained were evaluated by a rapid screening method on the agarized nutrient broth rollers with sulfur sources. The surface-grown columns were agar-coated with the test micro-organism Alcaligenes faecalis ATCC 8750, specific for cephalosporin C.
The larger diameter inhibitory zone isolate on agarated sodium thiosulfate was further tested in flask fermentation and in laboratory fermenters. The isolate was labeled with 188 VULM, cephalosporin C activity in the fermentation filter filtrates was determined to be 245153 in the 168th culture hour by spectrophotometric method / Claridge CA, Vaughan RW:
Antimicrob. Agents Chemother., 1969, 131) and by HPLC (prepared by WRI). The genealogical scheme of the strain is designated Scheme 1. Xzolate 188 VULM is stored in Cs. collection of microorganism UJEP in Brno under number CCM F-729.
Serving a Chemical Mutagen.
Kmeft OP 163 WIS was deposited in the form of a lyophilized can or short-lived agar / sporulation pod designated NB-II; the composition below. An aqueous suspension of 10 to 10 ® conidia / ml was prepared from slant. The suspension was filtered through a cotton plug. The N-nitrosomethylbiuret mutagen was added at a concentration of 0.4 to the suspension! conidia.
The mutangen was in contact with the culture for one hour on a shaker / 4Hz at 27 ° C. Dilutions of 10 µl to 10 conidia / ml were prepared from the mutangen-culture mixture after exposure. Petri dishes with agar NB-II agar were inoculated with the diluted suspensions. As a control, the spore suspension served without addition of the mutangene.
Mutangene operations were without daylight. Co-operation at Petrihomiskas lasted 11 days at 27 ° C. Individual colonies were transferred to angular agarNB-II and assayed for production activity on flasks. Activity of Physical Mutangene: A suspension of conidia was prepared from a culture on slanted agar by washing the surface of 10 ml of sterile water. The suspension was filtered through a cotton wool filter; dilutions of 10 to 10 conidia / ml were injected from the base slurry to inoculate a Petri dish (NB-II pod). The source of radiation was the germicidal OV-lamp / Philips TUV 30W, the wavelength of 253.7 nm is shared by the total spectral energy of 93.5%.
The conidia coated agar surface was exposed for 30 minutes at a distance of 30 cm. After irradiation, the Petri dishes were placed in a thermostate where the culture sporulated for 11 days at 27 ° C. Spores were transferred from the grown colonies by needle technique to the surface of the agar rollers.
After 5 days, the surface of the blocks was covered with culture. The blocks were placed on a glass agar plate containing 25% of Alcaligenes faecalis ATCC 8750 culture. Incubation blocks were maintained at 37 ° C for 18 hours. Evaluation of isolates was based on measurement inhibition zones around the blocks. The best isolate was from the aqueous colony to Petri dish-seeded on NB-II sloping agar and tested in both laboratory and quarter feed fermentations.
Agar Stage NB-II w / v Basto-beef Extract 0.3 Bacto-Peptone 0.5 Glucose 1.0 FeSO4.7H2O 0.005 MgSO4-7H2O 0.005 DL-Methionine 0.005 L-Cysteine 0.005 Agar Difco 2.5 pH Podes before Sterilization -6.8.
245153 4 Preparation of vegetative inoculum:
500 ml flasks were used, filled with 60 ml of inoculum, composed of: sucrose 1.5% w / v. corn extract 0.5 ammonium acetate 0.5 soybean oil 0.2 pH before. sterilization is adjusted to 7.0.
For inoculation, a culture of oblique agar or barley grains, so-called barley preserves, is used. The preparation of the vegetative inoculum takes 72 hours at 27 ° C on a shaking machine (4 Hz). Vegetative inoculum is inoculated with production batches filled in 500 ml flasks of 40 liters: cornstarch maize flour saccharose and glucoseCaCO3
Na2S2 ° 3-5H2 ° borax cachalot oil 5.0% w / v7.00,51,250,50,90,052,8
8% v / v is used for vaccination. vegetative inoculum. The cultivation has the same conditions as the preparation of the vegetative inoculum, the cultivation time of 168 hours. Comparison of the production activity of the Cephalosporium acremonium WIS OP-163 strain with the 8c VULM and 188 VULM isolates is shown in Table 1. The results obtained in the flask fermentation are compared:
Table 1 experiment number strain prod. activity / yg / ml / CFS-C 1 WIS OP-163 350 8c VULM 1,860 2 8c VULM 1,930,188 VULM 4,000 3,188 VULM 4,000 9 VULM 1,680
Strain used in fermentation using DL-methionine as a source of sulfur. In Laboratory Glass Dancers / Ex. the CSAV workshop /, volume 2 liters, working volume 1 liter, was under conditions: aeration 0.75 volume air / volume of the batch, mixing 10 Hz, temperature to 24 cultivation hours 28 ° C, then to the end of fermentation 23 ° C, achieved by with an activity of 5,200 µg / ml in 168 hours of culture.
Tribal Morphology:
The morphology of the Acremonium chysogenum CCM F-729 strain can be described as follows: In mono-colony sieving, the creamy center of the beige-colored colony is 5 to 7 mm in diameter. The colony's surface is wrinkled, the edges radially grooved, the center of the colonies elevated
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CS968281A CS245153B1 (en) | 1981-12-23 | 1981-12-23 | Microorganism strain Acremonium chrysogenum |
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CS968281A CS245153B1 (en) | 1981-12-23 | 1981-12-23 | Microorganism strain Acremonium chrysogenum |
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CS245153B1 true CS245153B1 (en) | 1986-08-14 |
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CS968281A CS245153B1 (en) | 1981-12-23 | 1981-12-23 | Microorganism strain Acremonium chrysogenum |
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1981
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