Produkcija i karakterizacija enzima inulinaza Aspergillus spp. za dobijanje fruktooligosaharida

Abstract

Ova disertacija se bavi ispitivanjem mogućnosti primene izolata gljive roda Aspergillus spp. za produkciju enzima inulinaznog kompleksa u cilju dobijanja fruktooligosaharida. Razvijen je brz i pouzdan difuzioni test za detekciju proizvođača inulinaznog kompleksa korišćenog u ispitivanju 39 izolata vrste roda Aspergillus, prethodno identifikovanih do nivoa vrste, umnožavanjem CaM gena. Izolati su ispitani na mogućnost produkcije mikotoksina na genetskom nivou umnožavanjem biosintetskih klastera gena za fumonizin i ohratoksin, a analitičkim metodama ispitano je prisustvo mikotoksina u enzimskom preparatu. Ukrštanjem rezultata difuzionog enzimskog testa i mogućnosti produkcije mikotoksina odabran je soj identifikovan kao Aspergillus welwitschiae FAW1, koji ne produkuje mikotoksine i potencijalno je dobar proizvođač enzima za dobijanje FOS-ova, što ga čini pogodnim za upotrebu u proizvodnji hrane. Inulinazni enzimski kompleks je produkovan tokom fermentacije gljive na čvrstoj podlozi upotrebom različitih inducibilnih supstrata (tritikale, jerusalimska artičoka i pšenične mekinje). Svi enzimi su okarakterisani i dobijene su različite aktivnosti u zavisnosti od supstrata: egzoinulinazna – InuE (2,4 U/mL) i endoinulinazna – InuA (34 U/mL) dobijene su na tritikaleu, dok je najveća β-fruktofuranozidazna – FFase (6,3 U/mL) i fruktoziltransferazna – FTase aktivnost dobijena na podlozi sa jerusalimskom artičokom, pšeničnim mekinjama i peptonom. Koprodukcijom inulinaznih enzimskih kompleksa pokazano je da se ista gljiva može koristiti za obe metode dobijanja FOS-ova (FOSs i FOSh) u zavisnosti od podloge na kojoj 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 za dobijanje 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. Dobijeni FOSs i FOSh poseduju značajan antioksidativni potencijal što ih čini dobrim kandidatima za dodatak funkcionalnoj hrani.

This dissertation examines the possibility of using isolates of Aspergillus spp. for the production of the inulinase complex enzyme for obtaining of fructooligosaccharides. A rapid and reliable diffusion test was developed for the detection of the inulinase complex enzyme producers used in the examination of 39 Aspergillus spp. isolates from the black aspergilli group, previously identified to the species level, by amplification of the CaM gene. The isolates were examined for the possibility of mycotoxin production on the genetic level by amplifying the biosynthetic gene clusters for fumonisin and ochratoxin. The presence of mycotoxins in the enzyme preparation was examined using analytical methods. By crossing the results of the diffusion enzyme test and the possibility of mycotoxin production, a strain identified as Aspergillus welwitschiae FAW1 was selected as a potentially good producer of enzymes for obtaining FOS, which does not produce mycotoxins what makes it safe for use in food production. The inulinase enzyme complex was produced during the fermentation of the fungi on a different solid inducible substrates (triticale, Jerusalem artichoke and wheat bran). All enzymes were characterized and different activities were obtained depending on the substrate: exoinulinase - InuE (2.4 U/mL) and endoinulinase - InuA (34 U/mL) were obtained on triticale, while the highest β-fructofuranosidase - FFase (6.3 U/mL) and fructosyltransferase - FTase activity were obtained on a medium with Jerusalem artichoke, wheat bran and peptone. The co-production of inulinase enzyme complexes showed that the same fungi can be used for both methods of obtaining FOS (FOSs and FOSh), depending on the substrate on which it was grown. A new zymographic method for the simultaneous detection of enzymes InuA, InuE and 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 the A. welwitschiae FAW1 genome, which is considered responsible for the expression of FTase and FFase enzymes, was confirmed. The obtained FOSs and FOSh have significant antioxidant potential, which makes them good candidates for use in functional food.