Characterization Of The Coelomic Fluid Proteases Of Sea Cucumber Stichopus Japonicus And Their Effects On Autolysis

Sea cucumber has strong autolysis capabilities,which is a troublesome problem during fishing and processing.The chemical composition of sea cucumbers rapidly changes postmortem due to autolytic processes.Coelomic fluid(CF)is considered to pass aperture and percolating through the body wall,and might play a role in autolysis of sea cucumber.The present study was conducted to investigate the characterization of the coelomic proteases and their effect during sea cucumber autolysis.So evidences showed in this paper may further advance our understanding of the mechanism of autolysis.They may be well applied in storing and processing of sea cucumber.CF was obtained from sea cucumber Stichopus japonicus,and the proteases in the supernatant after centrifugation is characterized.Optimum pH and temperature of the primary proteases in CF were determined using azocasein as substrate.Specific protease inhibitors(EDTA,PMSF,ABESF,TPCK,TLCK)and the effect of various metal ions(Ca2+,Mg2+,Cu2+,Mn2+,Zn2+)were studied.There exist proteases with optimum activities at pH3.5 pH7.5,pH9.0,pH 10.0,pH 11.5.Maximum activity was detected during temperature range from 40 to 50 ℃.The proteases are markedly inhibited by EDTA,then PMSF.Zymography was carried out to further analyze coelomic proteases.CF gave mainly two bands on the polyacrylamide gel using casein as substrate and gave more than six bands using gelatin as substrate.Gelatin-zymography result demonstrated inhibition ratio:EDTA>PMSF>TPCK>TLCK,indicating the involvement of metalloprotease and serine proteinase.TCA soluble oligopeptides analysis and SDS-PAGE was performed to investigate the effect of CF protease on tissue degradation of sea cucumber(outside,middle,inside).Sea cucumber tissue demonstrated distinctive degradation and content of TCA soluble oligopeptides increased after three hours incubation with sea cucumber tissue.All prompt the proteases in the CF have promoting effect during sea cucumber analysis.