## 2 Results
### Analysis of the \(\beta\)-Fructofuranosidase Activity Expressed in P. pastoris  
Biochemical characteristics of the \(\beta\)-fructofuranosidase RdINV expressed in the heterologous system were compared with those shown by the protein obtained from _R. dairennesis_. Proteins were purified and their activities at different pH and temperature values determined using sucrose as substrate. As previously published, the enzyme produced by _R. dairennesis_ displayed maximum activity at pH 5.0 and 60 \({}^{\circ}\)C, retaining \(\sim\)35% of activity at 70 \({}^{\circ}\)C [26]. In contrast, the heterologous protein showed it at pH 5.0 and 65 \({}^{\circ}\)C, retaining \(\sim\)50% of activity at 80 \({}^{\circ}\)C (data not shown). The hydrolytic activity was also evaluated using  
Figure 3: SDS-PAGE analysis of the purified \(\beta\)-fructofuranosidase RdINV and the deglycosylation treatment. **(a)** RdINV expressed in _R. dairennesis_ (lane 1) and _P. pastoris_ (lane 2). **(b)** The purified heterologous protein (lane 2) was treated with PNGase F (500 U) for 2 h (lane 1) or with mannosidase (2 U) during 1, 3, 8, 20, 48, and 72 h (lanes 3–8, respectively). **(c)** The heterologous protein (lane 1) was treated with PNGase F for 2 h (lane 2) and then with mannosidase for 7 h (lane 3). The positions of the molecular mass markers are indicated on the right in kDa. The theoretical molecular weight of PNGase F is 36 kDa and that of mannosidase 110 kDa, with two subunits of 66 and 44 kDa. Figure 2: Time course of the \(\beta\)-fructofuranosidase produced in _P. pastoris_, including construction cRdINV-plB4. **(a)** Yeast was cultured in 1-L flasks containing 200 mL of BMM (full circles), and extracellular activity (empty circles) was analyzed at the indicated times using sucrose as substrate. Each point represents the average of three independent measurements, and standard error are indicated. **(b)** Analysis on SDS-PAGE of the \(\sim\)170 kDa protein detected at the times culture above was analyzed (lanes 1–9: 0, 7, 21, 27, 46, 53, 70, 77 and 96 h, respectively). Numbers on the left indicate the positions of molecular mass standards (lane M) in kDa. different sized substrates (Table 1). Regardless of the producing yeast (_R. dairenensis_ or _P. pastoris_), the enzyme hydrolys sucrose, raffinose, 1-kestose, inulin, and nystose, but not substrates such as melibiose, lactose, and lactulose. Additionally, it showed very similar \(K_{\text{m}}\) values for sucrose and 1-kestose, but the catalytic efficiency was two times higher for the one expressed in _R. dairenensis_ compared to the heterologous enzyme (Table 2). The transfructosylating activity of the protein expressed in _P. pastoris_ was evaluated using sucrose as substrate. As expected, different FOS were detected in the reaction mixture, 6-kestose being the major transrtucosylation product. Additionally, neokestose and 1-kestose, as well as the tetrasaccharides neonystose and nystose and the disaccharide blastose, were produced (Figure 4a). Similar chromatographic profiles were previously obtained with the enzyme expressed in _R. dairenensis_, but some of the products could not be identified because the corresponding standards were not available. In that case, \(\sim\)68 and 11 g/L of 6-kestose and neokestose, respectively, were produced with a sucrose conversion close to 75% (w/w). The maximum concentration of FOS obtained in this work with the heterologous enzyme was 82.2 g/L (of which 6-kestose: 48.4 g/L; blastose and neokestose: 13.9 g/L each; neonystose: 3.1 g/L; 1-kestose: 2.9 g/L), representing \(\sim\)14% (w/w) of the total carbohydrates in the reaction mixture, and was reached in 9 h, with a total sucrose conversion close to 80% (Figure 4b). A blastose increase concomitant with the decrease of neokestose was obtained after 11 h reaction, where neokestose reached 10.9 g/L (\(\sim\)22% reduction), neonystose 3 g/L, and blastose 15.9 g/L (\(\sim\)14% increase). At this point, 41.7 g/L of 6-kestose (\(\sim\)14% reduction) and 1.3 g/L of nystose were quantified.