Characterization of proteases from the digestive tract of sea cucumber (Stichopus japonicus): High alkaline protease activity

Proteases from the digestive tract of sea cucumber (Stichopus japonicus) have been characterized by biochemical and electrophoretic techniques. Casein digestion assay revealed acidic proteases with optimum activity at pH 2.0 and pH 5.0, and alkaline proteases with optimal activity at pH 8.0 and 13.5, respectively. The influence of temperature and pH on the activity of these proteases was studied. Specific protease inhibitors and the effect of various metal ions were also studied. Copper, Ca2+ and Mg2+ renatured the ethylenediamine tetraacetate (EDTA)-denatured protease at optimal pH range of 12.0-14.0. Non-dissociating discontinuous polyacrylamide gel electrophoresis (PAGE) and sodium dodecyl sulphate-polycrylamide gel electrophoresis (SDS-PAGE) using casein as substrate with or without specific protease inhibitors confirmed the existence of at least three proteases, whose molecular weights were defined as 20.6, 39.1 and 114.1 kDa, respectively. Furthermore the 20.6 kDa protease with good pH and thermal stability was confirmed to be a metallo-protease containing Cu2+ and the 39.1 kDa protease, a serine protease, probably a collagenase.