## 3 Results and Discussion
### Detranase Production at Shaker Scale by K. phaffii DEX49A\(-\)Asp-\(\Delta\)N30  
Growth and enzyme secretion for _K. phaffii_ DEX49A\(-\)ASP-\(\Delta\)N30 clone were studied in triplicate for 48 h in 100 mL of the YPCGlycerol culture medium (Figure 3). The dried cell weight (DCW) reached 19 g\(\cdot\)L\({}^{-1}\) at 48 h.  
The product formation (\(r\)-\(Tm\)DEX49A\(-\)ASP-\(\Delta\)N30) curve measured as a function of enzymatic activity describes a continuous increase from the beginning to 48 h cultivation, which is typical of constitutive production [38]. The level of enzyme activity or amount of dextranase in the extracellular medium (273 U\(\cdot\)mL\({}^{-1}\) or 273 mg\(\cdot\)L\({}^{-1}\)) of _K. phaffii_ DEX49A\(-\)ASP-\(\Delta\)N30 is higher than the 37-98 mg\(\cdot\)L\({}^{-1}\) reported by [PERSON] et al. [15] for \(r\)-\(Tm\)DEX49A in a similar _K. phaffii_ system at shaker scale. Although, in this case, the expression of the \(r\)-\(Tm\)dex49A gene was carried out under the transcriptional control of the methanol-induced promoter (pAOX1) for 120 h in a different culture medium. The only report found on the constitutive expression of \(r\)-\(Tm\)DEX49A in _K. phaffii_ at shaker scale with a duration  
Figure 2: Detection of \(r\)–\(Tm\)DEX49A\(-\)ASP–\(\Delta\)N30 production in recombinant _K. phaffii_ clones. Secreted and cell-associated dextranase levels (**I** and **II**, respectively) of 28 different clones (1–10) and (A–Q). Dextranase levels of different clones are normalized to the biomass (OD). (**III**): SDS–PAGE of the culture supernatant of clones D (2) and M (3) stained with Coomassieie Brilliant Blue R-250, (1) molecular weight standard PiNK Prestained Protein Ladder (NIPPON Genetics Europe, Germany). (**) refers to highly significant differences (\(p<0.001\)) between clone D and the other clones. Significance (or \(p\) value) was obtained by applying the Student’s test. of 72 h of culture, but using the _Saccharomyces cerevisiae_ SUC2 signal peptide for secretion, yielded 13.8 U\(\cdot\)mL\({}^{-1}\) or 13.8 mg\(\cdot\)L\({}^{-1}\) (considering a specific activity of \(\approx\)1000 U\(\cdot\)mg\({}^{-1}\)) [13]. All examples explored at shaker scale were inferior to the enzyme activity of the clone in this study. Detection in Polyacrylamide Gels of the Shaker Produced r\(-\)TmDEX49A\(-\)Asp-\(\)\(\Delta\)N30  
The accumulation of dextranase in the supernatant fraction at different times of the culture was analyzed through SDS-PAGE (Figure 4I), and the enzymatic activity was detected by zymogram (Figure 4 II). A progressive increase in the intensity of a CBB-stained protein band was observed, which corresponds to the molecular size of the _r\(-\)Tm_DEX49A\(-\)\(\Delta\)SP-\(\Delta\)N30 enzyme (\(-\)64-67 kDa, considering N\(-\)glycosylation) [13; 15; 39]. The product present in the cell-free culture predominates in the analyzed samples (Figure 4I), coinciding with that reported by [PERSON] et al. [15] and [PERSON] et al. [13]. However, when a higher cell biomass is achieved, the secreted protein could suffer some proteolytic degradation, as at least two smeared protein bands with lower MW were detected (Figure 4I, lanes 4-8). Proteolytic action is due to environmental stress or nutrient deficiency [40]. The zymography (Figure 4 II) confirms the _r\(-\)Tm_DEX49A\(-\)\(\Delta\)SP-\(\Delta\)N30 activity in the culture supernatant. The clear bands where there was hydrolysis of the dextran blue have a size close to 130 kDa, and the thickness of the gel zone with transparency increases in the same way that the culture time increases. This is similar to the CBB-stained gel, but with the difference being that the enzyme migrated less. Other authors report the same MW (CBB staining and dextran blue zymogram) for recombinant dextranases expressed in _Escherichia coli_[31; 41]. Similar results were obtained from the analysis in gels for the DEX protein produced by _Aspergillus allahabadi_ X26 and _Catenowulum agarrorans_ MNH15 [35; 42].