## 3 Discussion  
The \(\beta\)-fructofuranosidase RdINV from _R. dairenensis_ produces sugars of \({}^{1}\)F, \({}^{6}\)F, and \({}^{6}\)G-FOS series simultaneously, a property not shared by other \(\beta\)-fructofuranosidases from _Rhodotorula_ spp. This fact, together with the large size of the N-deglycosated protein, \(\sim\)144 kDa, led us to address its molecular characterization. The gene _RdINV_ characterized here was responsible for the analyzed \(\beta\)-fructofuranosidase activity since the encoded amino acid sequence already contained all the peptides detected in the protein purified from _R. dairenensis_ and showed the consensus sequences of enzymes with proved fructosyl transferases activity. Indeed, enzymes containing the typical MNDPNG and ECP sequences have been classified as low-level FOS-synthesis enzymes, with FOS productions representing \(\leq\)20% of total sugars in the reaction mixtures [29], and RdINV could be included in this group. In addition, potential proteins showing high similarity to RdINV were also  
Figure 4: Transfructsylating reaction analysis catalyzed by RdINV. (**a**) HPLC chromatogram of the mixture obtained after \(11\) h reaction. Reaction conditions: 600 g/L sucrose at 60 \({}^{\circ}\)C. Peaks correspondence: (1) fructose, (2) glucose, (3) sucrose, (4) blastose, (5) neokestose, (6) 1-kestose, (7) 6-kestose, (8) neorsorsuge, and (9) nystose. (**b**) Total fructoligosaccharides (FOS) production vs. the hydrolyzed sucrose. (**c**) Evolution of the different FOS detected in the reaction. \begin{table}
\begin{tabular}{c c c c c} \hline \hline \multicolumn{2}{c}{**Monosaccharides**} & \multicolumn{2}{c}{**Disaccharides**} & \multicolumn{2}{c}{**Polyols**} \\ \hline \multirow{2}{*}{**Acceptor**} & **New** & \multirow{2}{*}{**Acceptor**} & **New** & \multirow{2}{*}{**Acceptor**} & **New** \\  & **Product a** & & & **Product a** \\ \hline Fructose & + & Trehalose & + & Erythrifucl & + \\ Galactose & - & Palatinose & + & Galacitol & + \\ Glucose & + & Lactose & + & Sorbitol & + \\ Xylose & - & Lactulose & + & Mannitol & + \\ Arabinose & - & Leucrose & + & Ribitol & - \\ Mannose & - & Maltose & + & Xylitol & - \\  & Melibiose & - & & \\ \hline \hline \end{tabular}  
* * +, transfructsylation for the new acceptor positive indicates that new peaks were detected in HPLC chromatograms.  
\end{table} 
Table 3: Fructosyl-acceptor specificity of the RdINV expressed in _P. pastoris_. found in the genome of _Rhodotorula_ sp. JG-1b (97% identity, 82% coverage; KWU45911) and _Rhodotorula graminis_ (57% identity, 76% coverage; XP_018268095). RdINV showed a large N-terminal extension (177 amino acids) not present in other \(\beta\)-fructofuranosidases from yeasts but also showing similarity to other putative GH32 proteins from _Rhodotorula_, such as _Rhodotorula toruloides_ (68.1% identity, 83% coverage; M7X5U7) and _Rhodotorula taiwanensis_ (70.5% identity, 70% coverage; A0A255B6I2). Functionality of the gene _RdINV_ was analyzed in _P. pastoris_, basically, because this yeast lacks \(\beta\)-fructofuranosidase activity, shows high secretion of heterologous proteins, and a priori can process gene introns from other eukaryotic organisms [34]. However, protein RdINV, including a potential signal peptide of 20 amino acids, was only secreted by transformants containing the characterized gene without introns, which produced about 25 U/mL of \(\beta\)-fructofuranosidase activity (Figure 2a), thus, improving the level of activity reached by _R. dairenensis_ cultures (1.9 U/mL) by about 13 times and reducing the protein purification process to a simple concentration step of a yeast extracellular medium. The high molecular mass of RdINV (\(\sim\)170 kDa) was clearly due to its high degree of glycosylation since, after treatment with \(\alpha\)-(1-2,3,6) mannosidase, it dropped to \(\sim\)80 kDa, which is a similar size to other fungi deglycosylated \(\beta\)-fructofuranosidases [16; 35; 36].