Study On Serine Proteinase From The Viscera Of Sea Cucumber (Stichopus Japonicus)

Sea cucumber (Stichopus japonicus) is traditionally regarded as a seafood delicacy and iswidely consumed in China, Japan, Korea and other Asian countries. In China, the total output ofsea cucumber production is increasing annually to meet consumer demand. However, seacucumbers are easily subject to autolysis, which is a major problem in transportation andprocessing. Autolysis, or self-digestion, is a common phenomenon that occurs in various aquaticproducts and some species of fish. After the death of these animals, biochemical reactionsresponsible for anabolism cease while endogenous proteinases involved in catabolism retain theirfunction. Major digestive organs secrete proteinases such as trypsin, chymotrysin and cathepsin.Among these proteolytic enzymes, serine proteinase has significant activity in the hydrolysis ofcollagen. In the edible portions of sea cucumbers, the highly insoluble collagen fibers accountfor about70%of total protein. As a result, collagen hydrolysis is likely the main factor in seacucumber autolysis.Considering the significant autolysis rate of harvested sea cucumbers, we propose that yetunidentified proteinases in the sea cucumber viscera may also play an important role duringcollagen degradation. Sea cucumber collagen hydrolysis patterns were examined using the crudeenzyme extraction from viscera of sea cucumber. It was indicated that serine proteinase from theviscera of sea cucumber could hydrolyze collagen effectively. Therefore, the involvement ofserine proteinase for sea cucumber antolysis was proposed.In the present study, a serine proteinase was isolated to high purity from sea cucumberviscera by DEAE-Sephacel ion exchange, Sepharcyl S-200HR gel filtration and Mini-Q ionexchange column chromatographies. Experimental results showed that the yield of serineproteinase was1.6%, and the purification fold was331.7. The molecular mass of SP wasapproximately34kDa on SDS-PAGE. Peptide mass fingerprinting revealed that the enzyme wasidentical to111amino acid residues with proprotein convertase subtilisin/kexin type9preproprotein from Stichopus japonicus. The enzyme exhibited highest enzymatic activity at pH6.0~9.0and at35~40°C using both gelatin zymography and fluorogenic substrates. Thedegradation of gelatin by the purified serine proteinase was strongly inhibited by serineproteinase inhibitors such as benzamidine and pefabloc SC, while no significant inhibitoryeffects were observed using other inhibitors (E-64、EDTA、chymostatin、Pepstatin A).Studying of the relation between collagen and purified serine proteinase showed that the purified enzyme can effectively hydrolyze collagen at37°C and even at4°C. In the presence ofserine proteinase, the β-and γ-chains of sea cucumber collagen were hydrolyzed after incubationat37°C for15min, and the original collagen bands (α, β and γ) completely disappeared after3hincubation. However, in the presence of serine proteinase inhibitors, collagen hydrolysis wascompletely suppressed when incubated with serine proteinase. Aspartic proteinase, cysteineproteinase and metalloproteinase inhibitors, on the other hand, did not show any inhibitoryeffects on collagen hydrolysis. It assured that the purified serine proteinase involved in theautolysis of sea cucumber, and play a key role on the autolysis of sea cucumber. Our presentstudy provides valuable information for further analysis of the relationship between serineproteinase and sea cucumber autolysis.