## 3 Discussion
The protein glycosylation profile is species-specific and important for the protein folding, which often is related to levels of protein secretion, stability, and/or activity [37; 38]. Changes in the glycosylation pattern may lead to increased activity and/or stability when expressed in _P. pastoris_[38; 39]. Accordingly, a different glycosylation pattern could also be responsible for variations of activity detected between enzymes produced in _R. dairenensis_ and _P. pastoris_ with the substrates tested in this work (Table 2), although the percentage of glycosylation was very similar in both cases (Figure 3). The transferase activity of RdINV was not substantially altered after the expression in _P. pastoris_, as the main FOS produced was 6-kestose, followed by neokestose, 1-kestose, and two tetrasaccharides (Figure 4) that could be identified as neonystose and nystose. Blastose was also detected in reactions, a disaccharide previously obtained as a secondary product when using sucrose and the mycelium-bound transructosylating activity of fungi, such as _Cladosporium cladesporoides_[40], and levansucrases from bacteria, such as _Zymomonas mobilis_[41]. It was also produced by \(\beta\)-fructofuranosidases from the yeast _Sch. occidentalis_ and _X. dendrohorus_ by direct fructosylation of glucose and hydrolysis of neokestose, respectively [34; 42]. Curiously, RdINV could produce blastose using both ways since its production increased in reactions supplemented with glucose (Table 3, Figure S2a) and based exclusively on sucrose when the amount of neokestose was reduced (Figure 4c), which would make RdINV the first yeast enzyme showing this ability. In addition, RdINV was capable to transfer the fructosyl moiety of sucrose to a new unit of fructose, forming two products (Table 3 and Figure S2a). Most likely, in one of them, the two fructose units should be linked by a \(\beta\)-(2-6) bond due to the preference of RdINV to form 6-kestose, a levan-type trisaccharide where fructose units are connected by this linkage. The possibility of using _P. pastoris_ cells to remove glucose [43] and fructose from the reaction mixtures is a very attractive possibility in order to obtain suitable FOS mixtures for diabetic patients, which we also intend to evaluate in the future. Moreover, some of the evaluated disaccharides were also fructosylated (Table 3): among them, palatinose and trehalose, which shared this characteristic with the \(\beta\)-fructofuranosidase from _X. dendrohous_, but not with that from _Sch. occidentalis_[44; 45]. RdINV also used different alditols as fructosylation acceptors, including erythritol and mannitol, which could improve their functional properties. These low-digestible molecules are considered a food supplement for people with diabetes and intestinal disorders, but they also could increase the bifidobacteria community in the human gut microbiome [46; 47]. Therefore, the broad acceptor promiscuity of RdINV would increase its biotechnological potential and make it interesting for food and pharmacological sectors. Structural research of RdINV will help to understand the specificity and particular activity of this enzyme and provide more information to enlighten the molecular mechanisms of the determinants responsible for fructosyltransferase active, with the subsequent possibility to synthesize new oligosaccharidesaccharides in a regioselective way.