The Study Of Expression And Characteration Of Lysozyme C-terminal Peptide From Stichopus Japonicus

Sea cucumber lysozyme(SjLys)belongs to i-type lysozyme and exerts broad spectrum antibacterial action against Gram-negative and Gram-positive bacteria.It is reported that the N-terminal of SjLys encodes the glycosidase activity,and the C-terminal encodes the isopeptidase activity.Analysis of the expression and characteration of rSjLys-C can lay the foundation for antibacterial action against bacteria and the non-enzymatic machanism.The engineering bacteria was induced to express the soluble rSjLys-C protein.Then rSjLys-C expresses by engineering bacteria was purified by the nickel ion affinity chromatography and the Sephadex G-25.The purified rSjLys-C protein was cut by enterokinase.The rSjLys-C peptide was used to test the antibacterial activity.At the same time,the minimum inhibitory concentration and antibacterial rate were determined.The enzymology characteristics and kinetic parameters of isopeptidase were also analysed.The results showed that the rSjLys-C was over-expression in a soluble form,and rSjLys-C is successful purified with the yield of 68.1%.The rSjLys-C was cut into two pieces by enterokinase,about 9 kDa and 17 kDa.In the antibacterial test,rSjLys-C displayed strong antibacterial activity against Gram bacteria.And the minimum inhibitory concentration to Staphylococctus aureus and Vibrio parahaemolyticus were about 3.14×10-7 mol/L and 1.57×10-7 mol/L,respectively.It also demonstrated that rSjLys-C had the isopeptidase activity,the optimal pH was 7,and the optimal temperature was 37?.The characteristic constant Km was 0.28 mmol/L,kcat was 0.012314 s-1 and kcat/Km was 44(s·mol/L)-1.Above results showed that rSjLys-C is a soluble antibacterial peptide and has the isopeptidase activity.Compared with two kinds of known i-type lysozyme,Tapes japonica lysozyme and destabilase lysozyme,rSjLys-C has higher catalytic efficiency on the substrate L-?-Glu-pNA.In the future,rSjLys-C can be widely used in many industries,such as aquaculture,feed industry,preservative and fresh-keeping,medicine and health care.