SYNTHESIS AND CHARACTERIZATION OF FRUCTOSYLTRANSFERASE FROM Aspergillus oryzae IPT-301 FOR HIGH FRUCTOOLIGOSACCHARIDES PRODUCTION

Abstract

Fructooligosaccharides (FOS) are mainly produced by microbial fructosyltransferases (FTase, E.C.2.4.1.9), and Aspergillus oryzae IPT-301 has shown high fructosyl transferring and low hydrolytic activities, which leads to high FOS production yields, but the main operating parameters for its best performance have been scarcely studied. Thus, this work aimed to evaluate the cellular growth, production and characterization of mycelial and extracellular FTases by Aspergillus oryzae IPT-301. Experimental design showed that the extracellular FTase performance was optimized (high transfructosylation activity and low hydrolytic activity) for reaction pH 5.5-6.75 and temperature of 45-50 °C and was fitted by the Michaelis-Menten model, while the mycelial FTase showed better performance at pH below 6.5 and temperature above 46 °C and was better fitted by the Hill model. The results obtained showed that the fungus represents a promising source for FOS production on a laboratorial scale.

β-Galactosidase-Producing Isolates in Mucoromycota: Screening, Enzyme Production, and Applications for Functional Oligosaccharide Synthesis

“…Accordingly, the crude β-galactosidase cocktails purified in our study may have the transfructosylation activity that was responsible for the presence of FOSs during reactions with the mixture of o NPG–sucrose. In addition, because nystose is a common indicator sugar of transfructosylation activity [ 54 , 56 , 57 ], the presence of this compound in the o NPG–sucrose system further strengthens the occurrence of the reaction. Nevertheless, our results highlighted promising transfructosylation action of both the L. ramosa and the R. pusillus enzyme cocktails, that, although we have not yet investigated such enzyme activities, may be attributed to FFase and/or FTase enzymes present.…”

Effect of temperature, pH and storage time on the stability of an extracellular fructosyltransferase from Aspergillus oryzae IPT-301

“…The initial pH of the culture medium was adjusted to 5.5 before sterilization. The flasks were inoculated with 0.5 mL of a previously prepared suspension of 10 7 spores mL -1 , and incubated in orbital shaker at 30 ºC, 200 rpm for 64 h (Cunha et al, 2019;Faria et al, 2021). At the end of fermentation, the culture broth was vacuum-filtered (Whatman nº1) and the resulting supernatant containing an activity of 12.34 ± 0.23 U mL -1 for the FTase from A. oryzae IPT-301 was employed for further studies.…”

“…The enzyme FTase (culture broth) was incubated in tris-acetate buffer at 0.2 mol L -1 and pH 5.5, in the absence of substrate, in a broad temperature range (30 ºC, 40 ºC, 50 ºC and 60 ºC). The samples were collected at specified time intervals (1 h, 2 h, 4 h and 8 h) and immediately cooled in ice bath for 5 min, and the residual activity was determined under standard conditions, according to Ottoni et al (2012), Cunha et al (2019) and Faria et al (2021). The experiments were performed in triplicate.…”

“…The transfructosylation (AT) and hydrolytic (AH) activities were determined according to Ottoni et al (2012), Cunha et al (2019) and Faria et al (2021). For this, 0.1 mL of the soluble enzyme (culture broth) was incubated with 3.7 mL of sucrose solution (47%, m v -1 ) and 1.2 mL of tris-acetate buffer at 0.2 mol L -1 , at pH 5.5.…”

“…Several studies have reported optimal parameters for the production of FTase from A. oryzae IPT-301 and the behavior of its enzymatic activity according to pH, temperature and sucrose concentration (Cunha et al, 2019, Ottoni et al, 2012Perna et al, 2018). Furthermore, Faria et al (2021) and Gonçalves et al (2020) reported the enzymatic activity and thermal and operational stability of A. oryzae IPT-301 FTases immobilized by adsorption and encapsulation, respectively.…”