Characterization Of Serine Proteases In The Body Wall Of Sea Cucumber Stichopus Japonicus And Their Relationship With Autolysis

Sea cucumber has a strong power of autolysis.Endogenous proteases are the main reason for the degradation of protein during autolysis.Serine protease distributed widely in animal body,and was reported to be involved in autolysis of shrimp,fish and their products.In this presentation,the serine protease in the body wall of sea cucumber was extracted and characterized,its relationship with autolysis was further studiedFirst,the general enzymatic properties of the serine protease,and the properties of elastase and trypsin were characterized,including optimum pH,optimum temperature,and the effect of metal ions and specific inhibitors on the activity of them.Results demonstrated there existed at least four kinds of serine protease in the body wall of sea cucumber,including elastase and trypsin-like protease.The activity of the above protease was inhibited significantly by PMSF,TPCK,TLCK,AEBSF,SBTI.Second,the relationship between serine protease and autolysis was investigated by the above five inhibitors.After incubation at 46 ? for 5h or 6h,protein with MW about 200kDa degraded obviously.So,the effect of the above inhibitors on the protein degradation after 5h or 6h of autolysis was investigated by quantifying the change of soluble protein and TCA-soluble peptide and SDS-PAGE.Result of SDS-PAGE showed the above inhibitors could inhibited significantly the degradation of large protein with MW about 200kDa,with a descending sequence of AEBSF,SBTI,TPCK,TLCK,PMSF.Moreover,the five inhibitors could also inhibit the increase of soluble protein and TCA-soluble peptide during autolysis;AEBSF demonstrated the significant effect,with inhibition ration of 52.57%and 32.95%on those two indexes,respectively.The serine protease activity was also decreased by the inhibitors using Boc-Phe-Ser-Arg-MCA as substrate.The above results suggested that there existed various serine protease,and they were involved in the protein degradation during autolysis of sea cucumber.So,serine protease was an important kind of autolytic enzymes.