diplomsko delo
Maša Strašek (Avtor), Hrvoje Petković (Mentor), Hrvoje Petković (Član komisije za zagovor), Martina Avbelj (Član komisije za zagovor), Polona Jamnik (Član komisije za zagovor), Mojca Narat (Član komisije za zagovor), Martina Avbelj (Komentor)

Povzetek

Tetraciklini (TC) so poliketidni naravni proizvodi, ki vključujejo številna klinično pomembna antibakterijska zdravila, kot je oksitetraciklin (OTC) - antibiotik širokega spektra, ki ga proizvaja pomemben industrijski mikroorganizem Streptomyces rimosus. Zaradi vedno večje potrebe po novih in cenejših terapevtikih, farmacevtska industrija stalno optimizira bioprocese in tako zmanjšuje stroške bioproizvodnje antibiotikov. To vključuje tudi izboljšanje postopkov ekstrakcije in čiščenja. Cilj dela je bilo ovrednotiti in preizkusiti različne postopke ekstrakcije tetraciklinov iz fermentacijske brozge kulture Streptomyces rimosus. Uporabili smo gensko spremenjeni sev bakterije S. rimosus, ki med biosintezo oksitetraciklina proizvaja dva tetraciklinska medprodukta – dehidro TC in dehidro OTC. Kot kontrolni sev smo uporabili S. rimosus, ki proizvaja OTC v visokih koncentracijah. Izvedli smo primerjalno analizo, tako da smo zasičili kulturo z uporabo NaH2PO4, in to primerjali z metodo, kjer smo pH spreminjali z dodatkom HCl ali NaOH in nato izvedli ekstrakcijo z etil acetatom v razmerju 1:1. V gojišče smo dodajali tudi dve vrsti amberlitov, da bi dosegli takojšnjo adsorpcijo tetraciklinov, proizvedenih med bioprocesom. Tetracikline smo nato eluirali iz amberlitov z metanolom. Zastopanost tetraciklinov smo v obeh fazah – vodni in organski, analizirali s tankoplastno kromatografijo, visokotlačno tekočinsko kromatografijo in masno spektrometrijo sklopjeno s tekočinsko kromatografjo. Za najučinkovitejšo metodo se je izkazal dodatek amberlitov XAD16N v brozgo in ekstrakcija tetraciklinov iz amberlitov z metanolom. To metodo smo tudi prenesli v večji volumen in ekstrakte koncentrirali z vakuumskim uparjanjem. Končni ekstrakti so vsebovali tarčne tetraciklinske medprodukte v koncentracijah 2 g/L dehidro TC in 0,4 g/L dehidro OTC.

Ključne besede

tetraciklini;ekstrakcija;Streptomyces rimosus;oksitetraciklin;sekundarni metabolizem;

Podatki

Jezik: Slovenski jezik
Leto izida:
Tipologija: 2.11 - Diplomsko delo
Organizacija: UL BF - Biotehniška fakulteta
Založnik: [M. Strašek]
UDK: 602.3:579.873.7:604.4:615.33(043.2)
COBISS: 31078403 Povezava se bo odprla v novem oknu
Št. ogledov: 407
Št. prenosov: 23
Ocena: 0 (0 glasov)
Metapodatki: JSON JSON-RDF JSON-LD TURTLE N-TRIPLES XML RDFA MICRODATA DC-XML DC-RDF RDF

Ostali podatki

Sekundarni jezik: Angleški jezik
Sekundarni naslov: Optimization of the tetracycline extraction process from culture Streptomyces rimosus
Sekundarni povzetek: Tetracyclines (TC) are polyketide natural products that include a number of clinically important antibacterial drugs, such as oxytetracyclin (OTC) - a broad-spectrum antibiotic produced by the important industrial microorganism Streptomyces rimosus. Considering ever-growing need for new and economically affordable therapeutics, the pharmaceutical industry is continously optimizing the bioproceses and thus reducing the cost of antibiotic production. This also includes extraction and purification processes improvement. The aim of this work was to evaluate and test different processes of extraction of tetracyclines from the fermentation broth of Streptomyces rimosus culture. We used a genetically modified strain of S. rimosus, which produces two tetracycline intermediates during oxytetracycline biosynthesis - dehydro TC and dehydro OTC. S. rimosus, which produces OTC at high yield was used as a control. We carried out comparative anaysis where saturation of the fermentation broth using monosodium phosphate was compared to the methods where pH was corrected by HCl or NaOH, followed by the extraction with ethyl acetate in a ratio of 1: 1. Two types of amberlite resins were also added to the culture to achieve immediate adsorption of the produced tetracycline intermediated during the biosynthetic process. Tetracyclines were subsequently eluted from the resin with methanol. The presence of tetracyclines in both phases - aqueous and organic, was analyzed by thin layer chromatography, high pressure liquid chromatography and mass spectrometry coupled by liquid chromatography. The addition of XAD16N amberlite resins to the fermentation broth and the subsequent elution of tetracyclines from resins with methanol proved to be the most effective method. This method was also transferred to a larger volume and the methanol extracts were concentrated by evaporation under vacuum. The final extracts contained the target tetracycline intermediates in concentration of around 2 g/L dehydro TC and 0,4 g/L dehydro OTC.
Sekundarne ključne besede: tetracycline;extraction;oxytetracyclin;secondary metabolism;
Vrsta dela (COBISS): Diplomsko delo/naloga
Študijski program: 0
Konec prepovedi (OpenAIRE): 2022-09-08
Komentar na gradivo: Univ. v Ljubljani, Biotehniška fak., Študij biotehnologije
Strani: VIII, 22 str., [3] str. pril.
ID: 12033061