2.4. Sodium dodecyl sulphate -polyacrylamide gel electrophoresis (SDS-PAGE) and Protein Zymography

SDS-PAGE electrophoresis analyses of the crude extract and purified aliquots of the enzyme were carried out using a 10 % polyacrylamide running gel in accordance with the method described by Laemmli [18] under denaturing conditions. The molecular standard (SDS-PAGE Molecular Weight Standards, Broad Range) from Bio-Rad Laboratories (Santo Amaro, Brazil) was composed of myosin (200 kDa), β-galactosidase (116.25 kDa), phosphorylase b (97.4 kDa), albumin serum (66.2 kDa), ovalbumin (45 kDa), carbonic anhydrase (31 kDa), trypsin inhibitor (21.5 kDa), lysozyme (14.4 kDa) and aprotinin (6.5 kDa). Gel samples run at 150 V for 100 min. The bands were developed by the silver nitrate staining process.

The proteolytic activity of the purified enzyme was confirmed by zymography analysis according to the method described by Egito et al. [19] under non-denaturing conditions. SDS-PAGE electrophoresis was performed on polyacrylamide gels containing casein (10 mg.mL⁻¹) as substrate. The purified sample was lyophilized and resuspended in Zymography Sample Buffer (Bio-Rad Laboratories, Santo Amaro, Brazil). After electrophoretic migration, the gel was washed with 2.5 % Triton X-100 for 30 min. at 4 °C. The hydrolysis reaction was performed inside the gel during incubation at 37 °C for 48 h in a 0.05 M Tris−HCl buffer pH 7.5. Subsequently, the protease were detected by the dye solution composed of ethanol 40 % (v/v), acetic acid 10 % (v/v) and 1 mg.mL^-1 of Coomassie Briliant Blue (Sigma Aldrich, St.Louis, USA) for 60 min. Soon after, a bleach solution composed of ethanol 30 % (v/v) and acetic acid 7.5 % (v/v) was added until translucent bands appeared.