Degradation Effects Of Trypsin On Collagen Macromolecules From Body Wall Of Sea Cucumber(Stichopus Japonicus)

Sea cucumber is a kind of important marine economic animal,and is very susceptible to external stimuli.In response to external stimuli,the body of sea cucumber relaxtion,mucoid degeneration take place,and part of the initially firm body wall even flow out in a sticky mass.Such massive tissue autolysis can lead to severe postharvest quality deterioration of sea cucumber,consequently cause heavy economic losses.The body wall of sea cucumber is a type of mutable collagenous tissues(MCT).MCT is composed of microfibrils and collagen fibers,including the basic structural units such as collagens,proteoglycans and glycoproteins.Microfibrils surround and separate collagen fibers and form networks,which are responsible for the mechanical stability of sea cucumbr body wall.Therefore,the dramatic changes in apparent appearance and mechanical properties of body wall of sea cucumber during autolysis must be due to the degradation of macromolecules.These years,researchers found that the endogeneous proteases of sea cucumber can degrade the collagen and no-collagenou proteins from sea cucumber.However,the action of serine proteinases on the structural changes of collagen fibers and microfibrils,as well as the major biologically relevant substrates in body wall of sea cucumber,is still unclear.In this study,collagen fibers were extracted from Stichopus japonicas body wall,which were used as a model to reveal the role of serine proteases in autolysis process of Stichopus japonicas body wall.The model was hydrolyzed by trypsin,a kind of serine protease,and the dynamic changes of MCT hierarchy structure were observed by using scanning electron microscopy(SEM),the changes of high-level structures of collagen were observed by using differential scanning calorimetry(DSC)and Fourier transform infrared spectroscopy(FTIR),and the changes in soluble components were analyzed by using sodium dodecyl sulphate polyacrylamide gel electrophoresis(SDS-PAGE),high-performance liquid chromatography(HPLC)and chemical analysis.The purpose is to reveal the role of serine protease in degradation of collagen fibers,the major collagen macromolecules in MCT.SEM data showed that collagen fibers were the primary structural units of the isolated sample,which could be used as a model to study the degradation effects of enzymes on collagen macromolecules.After 72 h of incubation,typsin caused partial disaggregate of collagen fiber into collagen fibrils on its surface.However,the main body of collagen fibers was still intact,indicating that typsin just led to slight degradation of collagen fibers.SDS-PAGE and chemical analysis indicated that typsin can progressively liberate total soluble substances,proteins,glycosaminoglycan and hydroxyproline from collagen fibers.After 72 h,the dissolution rate of total soluble substances,proteins and hydroxyproline as well as dissolution amount of glycosaminoglycan of typsin-treated sample was 2.70%,3.92%,0.21%,and 1515.30?g/g,respectively.While the corresponding values of total soluble substances,proteins and glycosaminoglycan for blank control sampe were 1.87%,0.81% and 530.66?g/g,respectively.Meanwhile,there was no detectable hydroxyproline in the soluble substances of blank sample.This indicated that typsin can cause slight damage to collagen fibers.FTIR analysis showed that the MCT model kept the characteristic amide-bands A,I,II and III of the basic structure of the collagen molecule.Typsin treatment did not cause the changes of FTIR spectrum of the MCT model,incicating no dramatic changes in the secondary structure of collagen happened.DSC analysis indicated that the collagens embedded within the higher-level structures such as collagen fibers and collagen fibrils were thermally more stable than the monomeric collagen.Typsin treatment did not cause the changes of thermal stability of the MCT collagen macromolecules,indicating no dramatic changes in the high-level structures of collagen happened.In summary,trypsin can cause slightly damage to MCT collagen macromolecules of collagen fibers.Therefore,it was speculated that serine protease may take part in the autolysis of sea cucumber,but it might be not play the dominant role in this process.