Study Of Proteases From Digestive Tract Of Sea Cucumber (Stichopus Japonicus)

The digestive proteases from digestive tract of sea cucumber (Stichopus japonicus) have been characterized by biochemical and electrophoretic techniques. Casein digestion assay revealed that there exist acidic proteases with optimum activities at pH 2.0 and pH 5.0, and alkaline proteases with optimal activities at pH 8.0 and 13.5 respectively in the digestive tract of sea cucumber. Then the influence of temperature and pH on the activities of these digestive proteases was studied. Further investigations were preformed by pre-incubating these proteases with specific protease inhibitors and various metal ions to confirm the characters of the proteases. Next 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 at a series of pH range confirmed the existence of at least three proteases. The molecular weights of them were defined to be 20.6, 39.1 and 114.1 kDa respectively. Most importantly, the 20.6 kDa protease with perfect pH and thermal stabilities is confirmed to be a metallo-protease and the 39.1 kDa protease is a serine protease, probably a callagenase. Then the high alkaline protease (HAP) with optimum pH of 13.5 was purified using a combination of ion exchange and gel filtration chromatography. The purified HAP was then characterized in detail. It has relative good thermal and pH stability with optimum pH of 13.5 and optimal temperature at 37. It can digest native collagen. According to inhibition profiles obtained with numerous specific protease inhibitors and metal ions, the HAP was confirmed to be a collagenolytic-metalloprotease containing Cu2+ with serine protease like activity. The Km of HAP was confirmed to be 3.89 g/L and it has more ot-helix than (3-sheet.