海参内脏胶原降解酶的纯化及性质研究

Study on proteinases responsible for collagen degradation in the viscera of sea cucumber(Stichopus japonicus)

针对海参自溶现象,研究海参内脏中与胶原降解相关蛋白酶的存在情况,以期了解内源性蛋白酶在海参自溶过程中发挥的作用,为解明自溶的内在机理提供理论依据。通过硫酸铵盐析、DEAE-Sepharose阴离子交换柱层析、Sephacryl S-200凝胶过滤柱层析、Phenyl Sepharose FF疏水柱层析及M ini Q离子交换柱层析等方法,从海参内脏中分离纯化出降解明胶的蛋白酶。利用荧光底物研究其酶学性质及其对海参体壁胶原蛋白的降解情况。结果表明,该酶最适温度为30℃,最适p H为7.5,可被金属蛋白酶抑制剂EDTA、EGTA完全抑制,亦可被PM SF、Pefabloc SC等丝氨酸蛋白酶抑制剂完全抑制。底物特异性表明,该酶只对金属蛋白酶荧光底物有分解作用,推测其为金属蛋白酶。该酶在4和37℃下均能有效地分解海参体壁胶原蛋白,表明其在海参自溶过程中起重要作用。

Sea cucumbers are easy to undergo autolysis after harvest or under physical stimulations.Proteinases in sea cucumber viscera are supposed to play important roles in the degradation of major structural protein collagen. In the present study,crude proteinases were extracted from sea cucumber viscera and their hydrolysis ability on gelatin was investigated by zymography. The degradation of gelatin by crude proteinases was strongly inhibited by serine proteinase inhibitor PM SF and metalloproteinase inhibitors,including EDTA and EGTA. It was of interest to notice that the gelatinolytic activity of an enzyme with molecular weight of 33. 2 ku was activated by Ca^2 ＋and inhibited by EDTA,indicating that it may belong to metalloproteinase. This proteinase was further purified to homogeneity from sea cucumber viscera by 80%ammonium sulfate fractionation and a combination of chromatographic steps including DEAE-Sepharose ion exchange,Sephacryl S-200 gel filtration,Phenyl Sepharose Fast Flow hydrophobic interaction and M ini Q Sepharose ion exchange. The optimum temperature and p H of the finally purified proteinase were 30 ℃ and7. 5,respectively. Its enzymatic activity was almost completely suppressed by metalloproteinase proteinase inhibitors（ EDTA,EGTA） and serine proteinase inhibitors（ PM SF,Pefabloc SC）. Furthermore,the enzyme effectively hydrolyzed native sea cucumber collagen both at 4 and 37 ℃,suggesting its important role in the autolysis of sea cucumber.