## 3 Results and Discussion  
r-TmDEX49A-\(\Delta\)SP-\(\Delta\)N30 Variant and Histochemical Screening of Dextranolytic Activity in K. phaffii Clones  
Figure 11 shows the _r-Tm_DEX49A-\(\Delta\)SP-\(\Delta\)N30 in pJexpress915 (ATUM, USA). _E. coli_ TOP10 was transformed with the _K. phaffii_ integrative expression vector pDEX49A-\(\Delta\)SP-\(\Delta\)N30. The expression module was detected in an agarose electrophoresis (Figure 11). The size pattern obtained in the electrophoretic assay after restriction enzyme treatments corroborates the presence of the desired genetic construct (Figure 11). The synthetic DNA fragment was verified by Sanger sequencing. The native strain K. phaffii PSS 9010 and the integrative expression vector pDEX49A-\(\Delta\)SP-\(\Delta\)N30 were selected as the expression system to produce and secrete the biologically active truncated dextranase. The expression vector harbors a mature region comprising the amino acids 51-608 of _r-Tm_DEX49A-\(\Delta\)SP-\(\Delta\)N30 fused in frame with the _Saccharomyces cerevisiae_ prepro \(\alpha\)-factor (MF\(\alpha\)-2) signal sequence under the constitutive pGAP and the AOX1 terminator. Transformation of PSS 9010 with the expression vector Swal-linearized yielded numerous transformants on the solid medium with dextran blue (YPDSDB) plates containing various concentrations of ZeocinTM to screen for potential multiple inserts. This histochemical screening system has been previously reported [33; 34; 35; 36]. Twenty-eight colonies showing the positive dextranase phenotype through the formationof degradation halos around the colony were selected for further analysis. The enzyme secreted by the producing clones diffuses into the solid medium and hydrolyzes the dextran blue, resulting in a radial halo of transparency (Figure 1 III,IV). The transformants D and M showing a higher radial halo of transparency on the YPDSDB plate containing 1000 \(\upmu\)g\(\cdot\)mL\({}^{-1}\) of Zeocin(tm) (Figure 1 IV) also outperformed the dextranase activity level in the shake flask cultures compared with the rest of the transformants. This phenomenon of clonal variation due to a different copy number of expression cassettes is common in strains of _K. phaffii_ genetically modified by inserting genes into the genome, as was reported for the expression of tetanus toxin fragment C [37]. Selection of the Best K. phaffii Clone Producer of \(r\)-TmDEX49A-\(\Delta\)SP-\(\Delta\)N30  
Dextranase activity was determined from the supernatant of the selected clones. The levels of enzymatic activity obtained are shown in Figure 2I,II. The supernatant and cell fractions of 28 selected transformants were analyzed. The dextranase activity in the culture supernatant represented 93% of the total enzyme activity, while only 7% was associated with the cellular fraction. The highest value of enzymatic activity was obtained from the supernatant produced by clone D with 257 U\(\cdot\)mL\({}^{-1}\), followed by clone M with 204 U\(\cdot\)mL\({}^{-1}\) (Figure 2 II). The specific activity of the truncated variant resulted in being nearly the  
Figure 1: General cloning and screening strategy to isolate dextranolytic _K. phaffii_ clones. Detection of the DNA fragment encoding \(r\)–TmDEX49A–\(\Delta\)SP–\(\Delta\)N30. (**I**)–Schematic representation of plasmid pDEX49A–\(\Delta\)SP–\(\Delta\)N30. (**II**)–DNA electrophoresis; lane 2: undigested plasmid pDEX49A–\(\Delta\)SP–\(\Delta\)N30, lane 3: pDEX49A–\(\Delta\)SP–\(\Delta\)N30/Swal, lane 4: pDEX49A–\(\Delta\)SP–\(\Delta\)N30/Swal–Pstl, and lane 6: purified DNA fragment of approximately 4.2 kb from sample of lane 4. Lanes 1 and 5: 1 Kb DNA Ladder RTU (Genetics, Japan). (**III,IV**)–Qualitative histochemical detection of dextranase activity on YPDSDB plates through the formation of a degradation halo around the colonies of the positive clones. YPDSDB plates supplemented with Zeocin 100 \(\upmu\)g\(\cdot\)mL\({}^{-1}\) (**III**) and 1000 \(\upmu\)g\(\cdot\)mL\({}^{-1}\) (**IV**).