Produkcija i karakterizacija enzima inulinaza Aspergillus spp. za dobijanje fruktooligosaharida
Production and characterization of inulinase enzymes from Aspergillus spp. for obtaining of fructooligosaccharides
Докторанд
Stojanović, SanjaМентор
Vujčić, ZoranЧланови комисије
Dojnov, BiljanaGavrović-Jankulović, Marija
Duduk, Bojan
Метаподаци
Приказ свих података о дисертацијиСажетак
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 upotr...ebom 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 fo...od 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.
Факултет:
Универзитет у Београду, Хемијски факултетДатум одбране:
05-06-2023Кључне речи:
inulinaze, jerusalimska artičoka, tritikale, Aspergillus spp., Aspergillus welwitschiae, mikotoksini, zimogram, fruktooligosaharidi, prebiotici, antioksidativnost / inulinase, Jerusalem artichoke, triticale, Aspergillus spp., Aspergillus welwitschiae, mycotoxins, zymogram, fructooligosaccharides, prebiotics, antioxidant activityRelated items
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