Characterizing Thermostable and Proteolysis-Resistant Alginate Lyase

The alginate lyase was biochemically characterized, specifically for thermostability and proteolysis resistance. The recombinant enzyme was purified through IMAC using gravity flow columns (His GraviTrap™, GE Healthcare, IL, USA), according to a previously described procedure^{29 (link),30} . The protein concentration was adjusted at 0.83 g/L for both assays. The thermostability analysis was performed as previously reported^{27 (link)}. The protein concentration in the recovered supernatant was quantified in triplicate using a NanoDrop 2000/2000c (NanoDrop Technologies; Thermo Fisher Scientific, Inc., Pittsburgh, PA, USA), and the results were validated through visualization of 14% SDS-PAGE gels, showing the intensity of the bands present in the supernatants. The gel images were acquired with BioRad ChemiDoc XRS imaging system (Bio-Rad, Hercules, CA, USA). The proteolysis resistance analysis was performed as already described^{27 (link)}. The alginate lyase was incubated with porcine pancreatin (VWR Chemicals, West Chester, PA, USA) or PBS solution and, afterwards, the samples were analysed by 14% SDS-PAGE and compared to a low molecular weight (LMW) protein marker (18.5 to 96 KDa) (Nzytech, Portugal) (Fig. 5). The resultant images were acquired with BioRad ChemiDoc XRS imaging system (Bio-Rad) and proteolysis was confirmed by visualizing fragments with different molecular weights.