Study on proteinases responsible for collagen degradation in the viscera of sea cucumber(Stichopus japonicus)

Sea cucumbers are easy to undergo autolysis after harvest or under physical stimulations.Proteinases in sea cucumber viscera are supposed to play important roles in the degradation of major structural protein collagen.In the present study,crude proteinases were extracted from sea cucumber viscera and their hydrolysis ability on gelatin was investigated by zymography.The degradation of gelatin by crude proteinases was strongly inhibited by serine proteinase inhibitor PMSF and metalloproteinase inhibitors,including EDTA and EGTA.It was of interest to notice that the gelatinolytic activity of an enzyme with molecular weight of 33.2 ku was activated by Ca²⁺ and inhibited by EDTA,indicating that it may belong to metalloproteinase.This proteinase was further purified to homogeneity from sea cucumber viscera by 80% ammonium sulfate fractionation and a combination of chromatographic steps including DEAE-Sepharose ion exchange,Sephacryl S-200 gel filtration,Phenyl Sepharose Fast Flow hydrophobic interaction and Mini Q Sepharose ion exchange.The optimum temperature and pH of the finally purified proteinase were 30 ℃ and 7.5,respectively.Its enzymatic activity was almost completely suppressed by metalloproteinase proteinase inhibitors(EDTA,EGTA)and serine proteinase inhibitors(PMSF,Pefabloc SC).Furthermore,the enzyme effectively hydrolyzed native sea cucumber collagen both at 4 and 37 ℃, suggesting its important role in the autolysis of sea cucumber.