Purification And Characterization Of Cathepsin L Enzyme From The Body Wall Of The Sea Cucumber Stichopus Japonicus

Sea cucumber is an important kind of aquatic and economic animal because of its abundant nutrition and bioactive substance. More attention has been paid to cultivation of sea cucumber and its medicinal purposes value. Many kinds of physics and chemical factors would cause sea cucumber autolysis. This autolytic phenomenon has developed into a major problem in fresh sea cucumber preservation, transport and product processing. The Studies have shown that this ability is common to many aquatic organisms and results from the high endogenous proteolytic activity in the body wall of the sea cucumber. So understanding the enzyme character of cathepsins will provide the important messages to control and utilize the autolysis of sea cucumber.In this article, cathepsin L was extracted, purified and its character was studied. The optimal extracting conditions of cathepsin L from body wall of the sea cucumber were ensured. Cathepsin L was purified through ion-exchanged chromatography and gel chromatography. The molecular weight of cathepsin L was measured through SDS-PAGE and character of the purified protease was studied.The results indicated that the best extracting condition of cathepsin L was50mmol/L pH5.0NaAc-HAc buffer containing5mmol/L cysteine,1mmol/L EDTA,0.2%Triton-100and degree of saturation80%of sulfate ammonium.The purity of purified protease was42.81fold than the original crude protease through the use of DEAE Sepharose CL-6B、Sephadex G-75and TSK-GEL HPLC. The molecular of the purified protease was63kDa through SDS-PAGE. Z-Phe-Arg-NMec was specific substrate. Km and kcat were69.92μM and12.80/S, respectively. The optimal pH and temperature of the purified protease were5.0and50℃, respectively. Thermal stability was below50℃. The protease was scarcely inhibited by Ca2+and Mg2+ions, moderately inhibited by K+, Mn2+and Fe2+ions and completely inhibited by Cu2+and Zn2+ions.Partial or complete inhibition was observed in the presence of thiol-blocking agents E-64and indoacetic acid, on the contrary thiol-activating agents DTT and EDTA enhanced the activity, which confirmed that the purified protease was a thiol-protease. Both serine-(PMSF, TI) and metallo-(1,10-phenanthroline) protease inhibitors were partially inhibited the activity. However, cysteine protease inhibitors, antipain and leupeptin evidently inhibited the purified protease. These results suggested that the purified protease belonged to cysteine protease and contained SH group(s). These results confirmed that the purified protease has the characteristic of cathepsin L and existed in form of its enzyme-inhibitor complex or precursor.