Study On Proteinases Responsible For Collagen Degradation In The Viscera Of Sea Cucumber (Stichopus Japonicus)

Sea cucumber is highly exploited and demanded in the Chinese market.However,Sea cucumbers are easy to undergo autolysis(melting)after long time away from seawater or under physical stimulations,particularly in the south of China,this restricts the development of sea cucumber.Collagen fibers are dominant components of the body wall of sea cucumber,suggesting the degradation of major structural protein collagen is the main reason for autolysis.In addition,abundant enzymes exist in sea cucumber while the proteinase responsibe for collagen degradation,however,remains unclear.Therefore,it is essential to elucidate the enzymatic softening mechanism of sea cucumber.Eversion of viscera is a peculiar physiological phenomenon of sea cucumber in response to environmental challenges.Thus,proteinases in the viscera of sea cucumber are proposed to play important roles in the degradation of major structural protein collagen.In the present study,the changes of tissue appearances in the process of UV-induced “melting” for sea cucumber(Stichopus japonicus)viscera were studied.Then,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 k Da 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 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 ion exchange.The optimum temperature and p H 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).In addition,another serine gelatinlytic enzyme was also purified to high homogeneity from the viscera of sea cucumber.The estimated molecular weight was 45 k Da,and the enzyme was active at temperature range of 35~40 ℃ under pH 8.0.The enzyme activity was completely inactivated by serine proteinase and metalloproteinase inhibitors.Divalent metal ions were not essential for the gelatinolytic activity,suggesting that it belongs to serine proteinase family.Furthermore,both the metalloproteinase and serine proteinase effectively hydrolyzed native sea cucumber collagen at 4 and 37 ℃,strongly suggesting their roles in the autolysis of sea cucumber.Concerning the rapid autolysis rate and its dependence on temperature,a variety of low levels of proteases may act synergistically.Along with eversion of viscera,proteinases in internal organs could contact to the body wall,even take part in collagen degradation.In this study,we purified and characterized two collagenolytic enzymes,which may enable us to gain a more complete understanding of the autolysis and find an appropriate way to keep quality and exploit them during the processing of sea cucumber.