## 2 Results  
### Molecular Characterization of the Gene RdINV and Analysis of the Amino Acid Sequence Encoded  
To isolate the gene encoding the \(\beta\)-fructofuranosidase from _R. dairenensis_, the enzyme was purified as referenced [26]. As expected, only a protein of \(\sim\)170 kDa was detected by SDS-PAGE, which was initially processed for amino acid sequencing using tryptic and chymotryptic digestion followed by MALDI-TOF-MS analyses. Protein retrieved the three tryptic peptides: PQVHYSPPK (526.8 m/z), PAASSSWGAENPFFTDK (906.4 m/z), and NPVLSVGSNQR (659.4 m/z) (Table S1) that already aligned with part of the sequences of \(\beta\)-fructofuranosidases from fungi as _Cordycys millitaris_ (ATY66193), _Aurebsidium melanogemun_ (ARG411451), and _Papilotrema aurea_ (AF084001), all included in the structural family GH32. In addition, this protein generated 14 chymotryptic peptides, three of them sharing part of their sequences with the referenced tryptic peptides (Table S1). By using primers directed to the peptide sequences characterized here (Table S2), a yeast genomic DNA sequence of 2559-bp was characterized, which included an open reading frame (ORF) of 2297-bp (potential gene _RdINV_) preceded by 129-bp. Two possible introns of 70 and 199-bp, flanked with the conserved sequence of yeast splicing sites, were also identified in the first half of the ORF and eliminated using specific primers.  
The gene characterized here encoded a polypeptide of 675 amino acids with a predicted molecular mass of 70.8 kDa, an isoelectric point of 4.5 units, a possible signal peptide for protein secretion of 20 amino acids, and 16 potential N-glycosylation sites. The probability of the protein O-glycosylation was also high considering that 72% and 62% of serine and threonine residues, respectively, could be glycosylated. All sequences found in the 170 kDa protein by MALDI-TOF-MS analyses (Table S1) were located in the encoded polypeptide. The overall deduced sequence of the potential protein RdINV was remarkably similar to proteins included in the family GH32, which had previously been functionally and structurally characterized. Furthermore, it was more similar to enzyme sequences from the ascomyce yeasts _S. cerevisiae_ (40% identity and 73% coverage) and _Sch. occidentalis_ (39% identity and 74% coverage), as well as _Aspergillus awamori_ (34% identity and 78% coverage) than to those from the basidiomycetes _X. dendorhous_ (28% identity and 27% coverage) and _Aspergillus kawachii_ (29% identity and 29% coverage).  
Figure 1 shows a structural alignment of the catalytic domains of RdINV and some structurally resolved proteins from eukaryotic microorganisms. All conserved motifs of the family GH32 were recognized in the RdINV sequence. Among them, the consensus sequences: WMNDPNG (FMNDPNG in RdINV), FRDP, and MWECPDF (AYECPNL in RdINV), including the catalytic triad (in bold), were identified as Asp188 (nucleophile), Asp312 (stabilizer of the transition state), and Glu362 (acid base catalyst in the hydrolysis mechanisms), respectively. A structural model of this protein based on the homologous exo-inulinase from _A. awamori_ AaEI [33], which showed the highest sequence coverage with RdINV, was obtained (Figure S1). This model displayed the characteristic bimodular architecture of the family GH32, a catalytic \(\beta\)-propeller, and \(\beta\)-sandwich domains linked by a short segment.