Purification And Enzymatic Characteristics Of Trypsin-like Enzymes From Euphausia Superba & Research On Its Biological Activities

Euphausia superba which belongs to Arthropoda Crustacea, is the key species to the Antarctic ecological system. It is rich in resources and has great potential for the development and use. Foreign studies show that the Antarctic krill proteinases is very effective in eliminating necrotic tissues and cells, treatment of corneal alkali burn and eliminating plaque etc. It has great medical purposes and 40% of protease activity comes from trypsin. Accordingly the purification of Euphausia pacifica's trypsin-like enzyme, the enzymatic characteristics study and the research on its biological activities has significant theoretic and practical importance.In this paper, The separation and purification of trypsin-like enzymes was conducted by saturated-precipitation method using 20-70% ammonium sulfate, Sephacryl S100 gel chromatography and DEAE Sepharose-F.F ion exchange chromatography. One component(TLEⅠ) was obtained and its molecular weight was about 30.0KDa. purification multiple was 3.19.The purified trypsin-like enzymes were active in extremely wide pH range of 3.0¹1.0 and the optimum values were between pH 7.0 and 9.0 at 37°C with BApNA as subtrate. And they were active in temperature range of 4⁸0°C, extremely wide, with optimum values between 30 and 50°C at pH 8.0, BApNA as subtrate. With metal ion, Mg²⁺,Ca²⁺ (0⁰.5 mmol/L) had activation on trypsin-like enzymes, Fe³⁺,Cu²⁺,Zn²⁺,Glycerine and Acetone had inhabitation on trypsin-like enzymes. Mn²⁺,Ethanol and Dimethyl sulphoxide(DMSO),activate trypsin-like enzymes, when their concentration is low, but they inhibit trypsin-like enzymes significantly in high concentration. the rabbit model of mechanical trauma was established with the crude enzyme extracted and pure enzymes compared with papain and saline to clean the wound. The result showed that crude enzyme and the pure enzyme had a very good result, crude enzyme was better.Diarrhea was induced in mice by cathartics (senna, rhubarb or 5-hydroxytryptamine(5-HT)). After 1h, the mouse were given by Antarctic krill proteinases at different doses(20mg.kg^-1& 40mg.kg^-1)while normal saline was negative control and smecta was positive control. The ratio of diarrhea and the accumulated times of diarrhea of mice were measured within six hours. The result showed that the Antarctic krill proteinases could decrease the times of diarrhea of mice induced by senna significantly, decrease the times of diarrhea of mice induced by rhubarb and 5-HT.Dextran sodium sulfate (DSS) was used to induce UC in mice and mice treated with Antarctic krill proteinases at different doses(20mg.kg^-1& 40mg.kg^-1)while normal saline was negative control and Sulfasalazine was positive control. The results of each group were compared by visual observation, EGF determination and histological observation. The result showed that the Antarctic krill proteinases could inhibite the reduction of mice weight, keep them in good spirits and reduce the phenomenon of blood in the stool. At the same time, the Antarctic krill proteinases could significantly improve the expression level of EGF in UC mice. It could also reduce the occurrence of ulcer symptoms, and the effect is proportional to its concentration.