<p>Zemljište predstavlja bogat izvor različitih mikroorganizama čiji<br />produkti metabolizma mogu biti od izuzetnog značaja za čoveka.<br />Dosadašnja ispitivanja mikrobnog diverziteta u zemljištu su<br />otkrila bogati biosintetski potencijal za proizvodnju novih<br />prirodnih proizvoda kod velikog broja mikroorganizama, naročito<br />kada je u pitanju klasa Actinobacteria. Među zemljišnim izolatima,<br />rod Streptomyces prednjači po broju identifikovanih bioaktivnih<br />molekula u odnosu na sve ostale bakterije. Stoga je jedan od<br />ciljeva u okviru ove doktorske disertacije izolacija streptomiceta iz<br />rizosfera medicinski značajnih biljaka sakupljenih na teritoriji<br />Republike Srbije (Papaver rhoeas, Matricaria chamomilla, i Urtica<br />dioica) i ispitivanje njihovog antifungalnog potencijala na različite<br />vrste kandida. Morfološki različiti izolati (ukupno 103) su izolovani<br />iz uzoraka rizosfera i okarakterisani kao streptomicete. Dve<br />različite podloge i dve procedure za ekstrakciju su korišćene da bi<br />se pospešila detekcija antifungalnih jedinjenja. Ispitan je uticaj<br />ukupno 412 ekstrakata na rast Candida albicans disk difuzionim<br />esejem pri čemu je utvrđeno da 42% (43/103) izolata imaju<br />sposobnost proizvodnje antifungalnih jedinjenja pri ispitivanim<br />uslovima. Pojedini ekstrakti su inhibirali rast važnih humanih<br />patogena poput Candida krusei, Candida parapsilosis, i Candida<br />glabrata. Na osnovu stepena i spektra antifungalne aktivnosti<br />devet izolata je odabrano za dalja istraživanja. Ispitana je<br />sposobnost njihovih ekstrakata da inhibiraju rast kandida u tečnoj<br />kulturi i u formi biofilma, a takođe je ispitan i njihov uticaj na već<br />formirane biofilmove kandide u koncentracijama od 8 do 250<br />pg/ml. Hromatografski profili ovih ekstrakata i uvid u njihovu<br />metaboličku raznolikost dobijeni su korišćenjem tečne<br />hromatografije visokih performansi. Tri ekstrakta sa specifičnom<br />antifungalnom aktivnošću podvrgnuta su hemijskim analizama s<br />ciljem da se detektuju i strukturno okarakterišu molekuli koji su<br />nosioci antifungalne aktivnosti. Na osnovu rezultata nuklearnomagnetno-<br />rezonantne spektroskopije otkriveno je da su aktivni<br />molekuli genistein, daidzein i staurosporin. Genistein i daidzein<br />koji su poznati fitoestrogeni poreklom iz sojinog brašna za koje je<br />poznato da inhibiraju ključne enzime u biosintetskom putu<br />steroida. Njihovo prisustvo je u ovom istraživanju detektovano<br />usled korišćenja sojinog brašna u hranljivoj podlozi. Kako<br />streptomicete u čijim ekstraktima su detektovani ovi molekuli<br />pokazuju sposobnost oslobađanja ovih važnih jedinjenja iz<br />kompleksne hranljive podloge, mogu se uzeti u razmatranje za<br />biotehnološku proizvodnju fitoestrogena. Staurosporin je<br />detektovan kao nosilac antifungalne aktivnosti kod ekstrakta soja<br />Streptomyces sp. BV410. Staurosporin je inhibitor protein kinaza i<br />njegovi derivati i analozi se koriste u kao antitumorski agensi.<br />Biosinteza ovog molekula je optimizovana do prinosa od 36,94<br />mg/l nakon 14 dana gajenja u hranljivoj podlozi koja sadrži<br />glukozu, skrob, manitol i sojino brašno (JS). Dalja optimizacija<br />hranljive podloge za biosintezu staurosporina ukazala je na<br />sledeći sastav hranljive podloge: 20 g/l glukoze, 0,36 g/l skroba,<br />21,46 g/l manitola, 17,32 g/l sojinog brašna. Primenom<br />definisanih optimalnih vrednosti i korišćenjem odgovarajućih<br />matematičkih modela, predviđeno je da će se na ovaj način<br />postići prinosi od 46,88 mg/l staurosporina i 12,05 mg/ml<br />biomase. Validnost predviđenih rezultata potvrđena je<br />izvođenjem bioprocesa u optimizovanoj hranljivoj podlozi (JSSta).<br />Ispitana je kinetika biosinteze staurosporina i produkcije biomase,<br />kao i potrošnje izvora ugljenika i razvijeni su odgovarajući<br />procesni modeli. Dodatna optimizacija je podrazumevala dodatak<br />suplemenata koji prema literature stimulišu sekundarni<br />metabolizam streptomiceta (joni cinka, gvožđa, fosfati, metil<br />oleat, ulje semenki grožđa). Ovi eksperimenti su izvođeni na tri pH<br />vrednosti (6,5, 7,5 i 8,5) a uspešnost bioprocesa je procenjivana 7.,<br />10. i 14. dana gajenja. Dodatna optimizacija je dovela do podatka<br />da dodatak soli gvožđa značajno pospešuje biosintezu<br />staurosporina sa povećanjem prinosa od 25%. Dobijeni rezultati<br />potvrđuju da su rizosfere medicinski značajnih biljaka značajan<br />izvor streptomiceta koje proizvode komponente sa<br />antifungalnom aktivnošću. Izolacija novog proizvođača<br />staurosporina i optimizacija procesa njegove biosinteze<br />omogućiće dalja istraživanja ovog jedinjenja koje može biti<br />osnova za razvoj novih antifungalnih i jedinjenja koja inhibiraju<br />angiogenezu. Rezultati dobijeni u okviru ovih istraživanja<br />predstavljaju početni korak ka potencijalnoj industrijalizaciji<br />proizvodnje staurosporina.</p> / <p>Different soils are still a source of remarkable microbial diversity<br />which also reflects in the unexplored chemical diversity. Recent<br />advances in assessment of microbial diversity from soil have<br />revealed the extraordinarily rich biosynthetic potential for the<br />production of new natural products among different microbial<br />strains, especially within the group of Actinobacteria. Among<br />bacterial soil isolates, representatives of Streptomyces genus are<br />the most prolific producers of bioactive compounds. One of the<br />objectives of the present study was to isolate Streptomyces spp.<br />from the rhizosphere soils of three ethno-medicinal plants<br />collected in Serbia (Papaver rhoeas, Matricaria chamomilla, and<br />Urtica dioica) and to screen their antifungal activity against<br />Candida spp. Morphologically different sporulating isolates (103<br />in total) were collected from rhizosphere soil samples and<br />determined as Streptomyces spp. Two different media and two<br />extraction procedures were used to induce the production and<br />facilitate identification of antifungals. Overall, 412 crude cell<br />extracts were tested against Candida albicans using disk<br />diffusion assays, with 42% (43/103) of the strains showing the<br />ability to produce antifungal agents. Also, extracts inhibited<br />growth of other important human pathogens: Candida krusei,<br />Candida parapsilosis, and Candida glabrata. Based on the<br />established degree and range of antifungal activity, nine isolates<br />were selected for further testing. Their ability to inhibit Candida<br />growth in liquid culture, to inhibit biofilm formation, and to<br />disperse pre-formed biofilms was assessed with active<br />concentrations from 8 to 250 pg/ml. High-performance liquid<br />chromatographic profiles of extracts derived from selected<br />strains were recorded, revealing moderate metabolic diversity.<br />The most potent extracts were subjected to comprehensive<br />identification and structural characterization of antifungal<br />compounds. Applying a bioactivity-guided isolation approach,<br />active compounds of three extracts were separated, and based<br />on NMR structure elucidation it was shown that active<br />compounds were genistein, daidzein and staurosporine.<br />Genistein and daidzein, soy phytoestrogens, are known to inhibit<br />key enzymes in the steroid metabolism pathway and were<br />coming from the fermentation medium containing soy flower.<br />Since isolated Streptomyces spp. showed good ability to extract<br />these molecules from complex medium, they can be further<br />considered for biotechnological production of these<br />phytoestrogens. One of the isolates, Streptomyces sp. BV410,<br />was characterized as an efficient staurosporine producer.<br />Staurosporine is a potent inhibitor of protein kinases and is<br />considered in anticancer therapy. The biotechnological<br />production of staurosporine by strain BV410 was optimized to<br />yield 36.94 mg/l after 14 days of incubation in soy flowerglucose-<br />starch-mannitol based fermentation medium (JS).<br />Further optimization of medium for biosynthesis of<br />staurosporine indicated the following optimal values of the<br />examined factors: the content of glucose of 20 g/l, starch 0.36<br />g/l, mannitol 21.46 g/l, soy flower 17.32 g/l. By applying the<br />defined optimal values and using the appropriate mathematical<br />models, the following responses were predicted: concentration<br />of staurosporine 46.88 mg/l and biomass yield 12.05 mg/ml. The<br />validity of the results was confirmed by performing the<br />biosynthesis of the staurosporine in the medium with optimal<br />composition (JSSta). Kinetics of staurosporine and biomass<br />production and carbon source consumption were examined and<br />process models were developed. Additionally, optimization of<br />staurosporine production was performed with different<br />supplements which, according to literature data, had stimulative<br />effect on secondary metabolism (Zn, Fe and P salts, methyl<br />oleate, grape seed oil). In order to improve the production of<br />staurosporine, effects of pH (6.5, 7.5 and 8.5) and incubation time<br />(7, 10 and 14 days) were also examined. It was found that<br />addition of FeS04 significantly improved the staurosporine yield<br />in comparison to the starting conditions (increase of 25%). Our<br />results proved that rhizosphere soils of ethno-medicinal plants<br />are a prolific source of streptomycetes, producers of compounds<br />with good antifungal activity. Isolation of the new staurosporine<br />producing strain, allowed for its detailed bioactivity assessment.<br />Staurosporine scaffold might serve as a lead structure for the<br />development of new antifungal and antiangiogenic agents. Also,<br />results obtained within this research represent the basis for the<br />further scale-up and potential industrialization of the proposed<br />production process.</p>
| Identifer | oai:union.ndltd.org:uns.ac.rs/oai:CRISUNS:(BISIS)111226 |
| Date | 13 December 2019 |
| Creators | Mojićević Marija |
| Contributors | Grahovac Jovana, Dodić Jelena, Cvetković Dragoljub, Vojnović Sandra |
| Publisher | Univerzitet u Novom Sadu, Tehnološki fakultet Novi Sad, University of Novi Sad, Faculty of Technology at Novi Sad |
| Source Sets | University of Novi Sad |
| Language | Serbian |
| Detected Language | Unknown |
| Type | PhD thesis |
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