Produkcija i karakterizacija enzima inulinaza Aspergillus spp. za dobijanje fruktooligosaharida

Abstract

Ova disertacija se bavi ispitivanjem mogućnosti primene izolata gljive rodaAspergillus spp. za produkciju enzima inulinaznog kompleksa u cilju dobijanjafruktooligosaharida. Razvijen je brz i pouzdan difuzioni test za detekciju proizvođačainulinaznog kompleksa korišćenog u ispitivanju 39 izolata vrste roda Aspergillus, prethodnoidentifikovanih do nivoa vrste, umnožavanjem CaM gena.Izolati su ispitani na mogućnost produkcije mikotoksina na genetskom nivouumnožavanjem biosintetskih klastera gena za fumonizin i ohratoksin, a analitičkim metodamaispitano je prisustvo mikotoksina u enzimskom preparatu. Ukrštanjem rezultata difuzionogenzimskog testa i mogućnosti produkcije mikotoksina odabran je soj identifikovan kaoAspergillus welwitschiae FAW1, koji ne produkuje mikotoksine i potencijalno je dobarproizvođač enzima za dobijanje FOS-ova, što ga čini pogodnim za upotrebu u proizvodnjihrane.Inulinazni enzimski kompleks je produkovan tokom fermentacije gljive na čvrstojpodlozi upotrebom različitih inducibilnih supstrata (tritikale, jerusalimska artičoka i pšeničnemekinje). Svi enzimi su okarakterisani i dobijene su različite aktivnosti u zavisnosti odsupstrata: egzoinulinazna – InuE (2,4 U/mL) i endoinulinazna – InuA (34 U/mL) dobijene suna tritikaleu, dok je najveća β-fruktofuranozidazna – FFase (6,3 U/mL) i fruktoziltransferazna– FTase aktivnost dobijena na podlozi sa jerusalimskom artičokom, pšeničnim mekinjama ipeptonom. Koprodukcijom inulinaznih enzimskih kompleksa pokazano je da se ista gljivamože koristiti za obe metode dobijanja FOS-ova (FOSs i FOSh) u zavisnosti od podloge nakojoj se uzgaja.Razvijena je nova zimogramska metoda za simultanu detekciju enzima InuA, InuE,FFase nakon jednog elektroforetskog razdvajanja enzima.Uspešno su prečišćeni glavni enzimi odgovorni za produkciju FOS-ova – InuA zadobijanje FOSh i FTase za dobijanje FOSs. Potvrđeno je prisustvo gena suc1 u genomu A.welwitschiae FAW1 koji se smatra odgovornim za ekspresiju enzima FTase i FFase. DobijeniFOSs i FOSh poseduju značajan antioksidativni potencijal što ih čini dobrim kandidatima zadodatak funkcionalnoj hrani.

This dissertation examines the possibility of using isolates of Aspergillus spp. for theproduction of the inulinase complex enzyme for obtaining of fructooligosaccharides. A rapidand reliable diffusion test was developed for the detection of the inulinase complex enzymeproducers used in the examination of 39 Aspergillus spp. isolates from the black aspergilligroup, previously identified to the species level, by amplification of the CaM gene.The isolates were examined for the possibility of mycotoxin production on the geneticlevel by amplifying the biosynthetic gene clusters for fumonisin and ochratoxin. Thepresence of mycotoxins in the enzyme preparation was examined using analytical methods.By crossing the results of the diffusion enzyme test and the possibility of mycotoxinproduction, a strain identified as Aspergillus welwitschiae FAW1 was selected as apotentially good producer of enzymes for obtaining FOS, which does not producemycotoxins what makes it safe for use in food production.The inulinase enzyme complex was produced during the fermentation of the fungi ona different solid inducible substrates (triticale, Jerusalem artichoke and wheat bran). Allenzymes were characterized and different activities were obtained depending on thesubstrate: exoinulinase - InuE (2.4 U/mL) and endoinulinase - InuA (34 U/mL) were obtainedon triticale, while the highest β-fructofuranosidase - FFase (6.3 U/mL) andfructosyltransferase - FTase activity were obtained on a medium with Jerusalem artichoke,wheat bran and peptone. The co-production of inulinase enzyme complexes showed that thesame fungi can be used for both methods of obtaining FOS (FOSs and FOSh), depending onthe substrate on which it was grown.A new zymographic method for the simultaneous detection of enzymes InuA, InuEand FFase after one electrophoretic separation of the enzymes was developed.The main enzymes responsible for the production of FOS were successfully purified -InuA for obtaining FOSh and FTase for obtaining FOSs. The presence of the suc1 gene in theA. welwitschiae FAW1 genome, which is considered responsible for the expression of FTaseand FFase enzymes, was confirmed. The obtained FOSs and FOSh have significantantioxidant potential, which makes them good candidates for use in functional food.