## 3 Results and Discussion
### Detranase Production at Shaker Scale by K. phaffii DEX49A\(-\)Asp-\(\Delta\)N30
The difference in apparent MW due to a delay in the _r\(-\)Tm_DEX49A migration in polyacrylamide gels containing dextran blue was recently suggested as the result of a tendency of this protein to form aggregates in the culture supernatant [13]. In this study, the phenomenon has been revisited, but the results point to other causes (see below, Section 3.7 and Figure 7). On the other hand, bands lower than 64-67 kDa (Figure 4I) did  
Figure 3: Cell growth and dextranase enzyme activity of _K. phaffii_ DEX49A\(-\)\(\Delta\)SP–\(\Delta\)N30. Abs\({}_{600}\), (DCW) values in g\(\cdot\)L\({}^{-1}\) and dextranase activity (U\(\cdot\)mL\({}^{-1}\)) of the supernatant in 100 mL of YPG medium. not appear to show dextranase activity (Figure 4 II), so they might be native proteins or inactive _r-Tm_DEX49A-ASP-AN30 degradation products. High Cell Density Fermentation of K. phaffii DEX49A-ASP-AN30 for High-Level Extracellular Production of _r-Tm_DEX49A-ASP-AN30  
The production of the _r-Tm_DEX49A-ASP-AN30 was carried out as a fed-batch culture in a 5 L bioreactor with the synthetic medium FM21-PTM1 [26]. The use of chemically defined or synthetic culture media in a large-scale fermentation strategy generally has the advantage of its greater batch-to-batch consistency compared to complex media, in addition to allowing for easy recovery of secreted protein in some cases [43], as well as the ability to reach up to 130 g:L\({}^{-1}\) of biomass in high cell density cultures [44]. In Figure 5, the cell growth and the secreted dextranase activity are shown. The fermentation strategy used in this study allowed for reaching DCW values above 100 g:L\({}^{-1}\) in 30 h of culture, without inhibiting cell growth or compromising the enzyme secretion. The enzyme activity values were compared with reports of dextranase production (_r-Tm_DEX49A) in _K. phaffii_ on a 5 L scale. In the first report of the recombinant production secreted dextranase (_r-Tm_DEX49A) in _K. phaffii_, it was possible to increase from 98 U:mL\({}^{-1}\) (98 mg:L\({}^{-1}\)) in the shaker (100 mL) to 3.2 g:L\({}^{-1}\) (\(\sim\)3200 U:mL\({}^{-1}\)) in a 5 L scale fermenter in 120 h and repeat methanol feedings for the induction of the AOX1 promoter, obtaining an enzymatic productivity of 26.6 mg:L\({}^{-1}\).h\({}^{-1}\)[15]. Subsequently, a new feeding strategy with methanol was proposed, where the best values of enzyme secretion were achieved for this system, reaching up to 5135 g:L\({}^{-1}\) (\(\sim\)5135 U:mL\({}^{-1}\)) in the culture supernatant, and a productivity of 42.7 mg:L\({}^{-1}\).h\({}^{-1}\) in a 2 L fermenter [45]. The values of the enzyme titer (3.2 g:L\({}^{-1}\) and 5135 g:L\({}^{-1}\)) exceed the ones reached in this study by two and three times, respectively, but in terms of the bioprocess system productivity, the outcome reported here for _r-Tm_DEX49A-ASP-AN30 is higher than for _r-Tm_DEX49A. This study also exceeds the recently reported values in a fermenter of 2 L (97.34 U:mL\({}^{-1}\) or 97.34 mg:L\({}^{-1}\)) and 30 L (62.64 U:mL\({}^{-1}\) or 62.64 mg:L\({}^{-1}\)) of _r-Tm_DEX49A produced in _K. phaffii_ in the optimized fermentation (100 h) methanol-induced manner with a productivity of 0.62 mg:L\({}^{-1}\).h\({}^{-1}\)[21]. Despite the wide use of the AOX1 promoter, it has some limitations, as methanol is a highly dangerous compound and even more so when working in large volumes, and storage can also be risky in industry [46,47]. Additionally, being a derivative from petrochemical sources, it may require purification steps to produce food and food additive products [48]. Such evaluations must be considered to design a technology in bioreactors and choose the most convenient system (pAOX1-inducible/pGAP-constitutive) that suits the needs. Figure 4: Analysis of the production of recombinant _r-Tm_DEX49A–ASP–AN30 in _K. phaffii_ at shaker level in 100 mL of YGlycerol medium. **(I)** SDS-PAGE of the fermented supernatant stained with CBB.