Study On Enzymatic Properties Of Cathepsin Family From Eriocheir Sinensis And Euhausia Superba

The Chinese mitten crab Eriocheir sinensis and Antarctic krill Euphausia superba are important crustacean species with high economic value in China.The crustaceans have no immunoglobulin in body fluids,and lacking immune-mediated immune response,but have congenital immunity defense system and can prevent the invasion of pathogenic microorganisms.Cathepsin is a class of lysosomal proteases,which is mainly divided into serine cathepsins(such as cathepsin A,CatA),cysteine cathepsins(such as cathepsin B,CatB;cathepsin H,CatH;cathepsin L,cat L),aspartic histamine cathepsin(cathepsin D,CatD),and involves in protein hydrolysis,innate immune defense response,antigen presentation,apoptosis and other physiological activities.In this study,we investigated the enzymatic properties of proteases by setting a series of factors(extraction time,pH,temperature,metal ions and activators and inhibitors)that affect the enzymatic reaction of E.sinensis cathepsins.(1)When the extraction time of hepatopancreas of E.sinensis was 120 min,the enzyme activities of CatA and CatD reached the highest,and the optimum extraction time of CatB,CatH and CatL respectively was 30 min,60 min,90 min.(2)The optimum pH for CatD,CatB,CatL and CatA was 5,and the optimum reaction pH for CatH was 7,indicating that cathepsin was mainly acid protease.(3)the optimum reaction temperature of CatA and CatB was 45 ?,the optimum reaction temperature of CatH and CatD was 55 ?,and the optimum reaction temperature of CatL was 65 ?,which indicated that CatL was high temperature resistant.(4)Mg2+had no obvious effect on CatA,and could inhibit the activation of CatB,CatH,CatL and CatD,while Ca2+,Mn2+,Pb2+,Cu2+ and Zn2+ inhibited CatA,CatB,CatH,CatL and CatD in different degrees.(5)DTT had an inhibitory effect on the activity of CatA and CatD,and had the activation on the activity of CatB,CatH and Cat L.EDTA,E-64 and leupeptin inhibited the activity of CatA,Cat B,CatH,CatL and CatD,the inhibition effect of E-64 and leupeptin on cysteine protease(CatB,CatH,CatL)was significant.(6)After the injection of Escherichia coli?Staphylococcus aureus and Saccharomycescerevisiae 12 h,the activity of CatB,CatH,Cat L and CatD from E.sinensis increased significantly,and the activity of CatA decreased significantly,indicating that CatA,Cat B,CatH,CatL and CatD participated in the autoimmune defense reaction.E.superba endogenous cathepsins could efficiently degrade proteins,cathepsins had a serious impact on the storage,transportation and preservation of E.superba.Some basic study on of E.superba cathepsins provide theoretical guidance for the storage,transportation and preservation of E.superba,and the application of cathepsins.So through the study of cathepsin different autolysis conditions of E.superba(autolysis time,autolysis pH,autolysis temperature,metal ions,activator and inhibitor)to explore the enzyme activity changes of CatB,Cat H,Cat L and CatD,so as to control its protein degradation.(1)After E.superba autolysis at 30 min,the residual enzyme activities of CatB and Cat D were higher;when autolysis was 60 min,the residual enzymes of CatH and CatL were higher(2)when E.superba autolysis buffer was at pH 5,the residual enzyme activities of Cat B was the highest;when E.superba autolysis buffer was at pH 6,CatH remained the highest enzyme activity;when E.superba autolysis buffer was at pH 4,CatL remained the highest enzyme activity;when E.superba autolysis buffer was at pH 8,the residual enzyme activity of CatD reached highest.CatB,CatH,CatL,Cat D were highly significant in each optimum autolysis buffer pH.(3)E.superba autolysis were at different temperatures(-20?,0?,20?,40?,60?),the residual enzyme activity of Cat B and Cat L activity is highest,when autolysis temperature is 0?;when autolysis temperature is 20?,the residual activity of CatD is highest;when autolysis temperature is 40?,the residual activity of CatL the highest.(4)CatB,CatH,and CatL are activated by E.superba autolysis buffer with 5 mmol/L Mg2+.Ca2+,Mn2+,Pb2+,Cu2+,Zn2+(5 mmol/L)had an inhibitory effect on them,and Mg2+ was significantly different from other metal ions.Mg2+ and Zn2+ had no significant effect on CatD,Ca2+,Mn2+,Pb2+,and Cu2+inhibited CatD,and the inhibitory effect of Cu2+was significant.(5)E.superba autolysis buffer with 2 mmol/L DTT and L-Cys could activate the activities of Cat B,CatH,CatL and CatD,activities of CatL and Cat D were inhibited by E.superba autolysis buffer with EDTA(1 mmol/L),E-64(0.1 mmol/L)and leupeptininhibitory peptide(0.1 mmol/L),E-64 and leupeptininhibitory peptide on cysteine protease(Cat B,CatH and CatL)inhibition was more significant.