## 3 Results and Discussion
### Detranase Production at Shaker Scale by K. phaffii DEX49A\(-\)Asp-\(\Delta\)N30
1—Molecular weight standard, PiNK Prestained Protein Ladder (NIPPON Genetics Europe, Germany); (2 to 8)—culture supernatants at hours 0, 4, 8, 12, 24, 36, 48. **(II)** Zymogram to detect dextranase activity; samples were analyzed in the same order from gel (A). As opposed to the induced systems, in this study, the product formation (_r-Tm_DEX49A-\(\Delta\)SP-\(\Delta\)N30) describes a continuous increase from the beginning to 30 h of cultivation, which is typical of constitutive production [49]. In the fed-batch culture, the feeding phase began at hour 14, taking as feeding criterion the carbon source depletion, judged by a sharp increase of dissolved oxygen as suggested by [PERSON] et al. [50]. In total, five increments were made between hour 14 and hour 24. The level of enzyme activity produced by _K. phaffii_ DEX49A-\(\Delta\)SP-\(\Delta\)N30 was 1614 U-mL\({}^{-1}\) (1.614 g\(\cdot\)L\({}^{-1}\)), which is the highest production level reported so far for a dextranase constitutively produced in _K. phaffii_. Recently, using a similar constitutive system, the dextranase _r-Tm_DEX49A was produced at 69 U-mL\({}^{-1}\) (69 mg\(\cdot\)L\({}^{-1}\)) in the culture supernatant using glycerol as a carbon source and 87 U-mL\({}^{-1}\) (87 mg\(\cdot\)L\({}^{-1}\)) and 110 U-mL\({}^{-1}\) (110 mg\(\cdot\)L\({}^{-1}\)) with molasses and glucose, respectively [13]. This same study reported a productivity of the constitutive _K. phaffii_ elite clone PpDEX(4x) grown with glucose (2157 U-L\({}^{-1}\)-h\({}^{-1}\) or 2.15 mg\(\cdot\)L\({}^{-1}\)-h\({}^{-1}\)), molasses (1706 U-L\({}^{-1}\)-h\({}^{-1}\) or 1.70 mg\(\cdot\)L\({}^{-1}\)-h\({}^{-1}\)), and glycerol (1353 U-L\({}^{-1}\)-h\({}^{-1}\) or 1.35 mg\(\cdot\)L\({}^{-1}\)-h\({}^{-1}\)) after 51 h of culture [13]. However, with the level of enzyme activity reported for _K. phaffii_ DEX49A-\(\Delta\)SP-\(\Delta\)N30 in this study, a productivity of 53,800 U-L\({}^{-1}\)-h\({}^{-1}\) (53.8 mg\(\cdot\)L\({}^{-1}\)-h\({}^{-1}\)) in 5 L fed-batch fermentation after 30 h using FM21-PTM1 culture media was reached. This outperformed at least 25-fold the productivity reported by [PERSON] et al. [13] and is, to the best of our knowledge, the highest productivity reported so far for a glycoside hydrolase family 49 dextranase. The results reported in this study confirm the efficiency of the constitutive pGAP to produce recombinant dextranase _r-Tm_DEX49A-\(\Delta\)SP-\(\Delta\)N30 using the _K. phaffii_ expression system. High yields of secreted enzyme in just 30 h of culture were obtained. Compared with the shake flasks batch cultures, the high cell density fermentation of _K. phaffii_, favorable dissolved oxygen levels, and nutrient balance in FM21-PTM1 culture medium in a 5 L fermenter all contributed to a significantly higher level of recombinant expression of the truncated dextranase variant _r-Tm_DEX49A-\(\Delta\)SP-\(\Delta\)N30. From the results of this study, it seems that the deletion of the first 30 amino acids in the dextranase _r-Tm_DEX49A-\(\Delta\)SP-\(\Delta\)N30 does not affect, and even improves, the production levels of the enzyme in the culture supernatant of _K. phaffii_ DEX49A-\(\Delta\)SP-\(\Delta\)N30. Therefore,  
Figure 5: Cell growth and dextranase enzyme production of _K. phaffii_ DEX49A–\(\Delta\)SP–\(\Delta\)N30 in a fed-batch culture. Abs\({}_{600}\), DCW values in g\(\cdot\)L\({}^{-1}\) and dextranase activity (U-mL\({}^{-1}\)) from the study in a 5 L fermenter using FM21–PTM1 medium. further studies are needed to corroborate this in more detail since the genetic constructions harbored by the clones used for the constitutive expression comparison were designed with different signal peptides. However, in addition, different strains of _K. phaffii_ (GS115 and PPS 9010) were used in both studies, and differences in the expression efficiency of dextranase genes could be associated with differences between the genetic background of the two strains [39].