Denaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE) was carried out as described by Laemmli
[19]. After electrophoresis, the gel was stained with Coomassie
Brilliant Blue R-250 dissolved in a water/methanol/acetic acid
(50:40:10) solvent for 1 h, then destained in the same solvent. The
zymogram was carried out according to Mitsunaga et al. [20]. After
electrophoresis, gel was incubated in a 1% soluble starch solution in
20 mM Tris–HCl buffer (pH 7.5) for 30 min at 37 ◦C, then discarded
the substrate solution and the gel was stained with a solution of
1.3% I2 and 3% KI for detection of activity.