Purification, Properties And Characterization Of Hatching Enzyme From Shrimp Penaeus Chinensis

During the hatching process of many animal species, a kind of protease, denominated as hatching enzyme (HE), plays an important role in digestion of the protective extracellular coats surrounding the embryos. HE, secreted at a specific stage by certain embryonic cells, provides a typical model in the studies of certain cell differentiation, specific protein synthesis, and special gene expression regulation during a certain stage of early embryos at the morphological and molecular level. It will be of great importance to understand its biochemical and enzymatic properties in the study and development of embryogenesis and embryo pharmacology. By using Penaeus chorion as a specific substrate, the hatching enzyme (HE) from Penaeus chinensis was purified by gel-filtration and ion-exchange chromatography, and characterized in terms of its molecular weight and enzymatic properties. Furthermore, use immunocytochemical staining and ultrastructure study to identify the appearance of HE and secretive cells in embryos of marine shrimp, Penaeus chinensis.By using Penaeus chorion as a specific substrate, the HE was prepared and purified by 67% ammonium sulfate precipitation, Sephacryl column gel-filtration, and DEAE-sepharose Fast Flow ion-exchange chromatography, and its biochemical and enzymological properties were identified in this study. Through purification, the Recovery of HE is 44.29%, the purification of HE is 48.05. It was found that the deduced molecular weight of HE in SDS-PAGE is about 43 kD, The Km value of the HE for casein was 7.47 mg ml-1. And its choriolytic activity was optimized at pH of 6.0 and at temperature of 40 oC, respectively. The HE activity was almost completely inhibited by SBTI, p-APMSF, bestatin and NEM, greatly inhibited by ovomucoid, TLCK, IAM, chymostatin and PMSF, and slightly inhibited by pepstatin A, TPCK, LBTI and leupeptin. And these results imply that HE is most probably a trypsin-type serine protease. Besides of these, the HE was extremely sensitive to EDTA, DETC, Zn²⁺, Ca²⁺, Mg²⁺ and Cu²⁺. These results imply that HE is most probably a metalloprotease. Combined with the results that the EDTA-pretreated HE activity could be perfectly recovered by Zn²⁺, it is indicated that shrimp HE is most probably a kind of Zn-metalloprotease.We use immunocytochemical staining and ultrastructure study to identify the appearance of HE and secretive cells in embryos of marine shrimp, Penaeus chinensis. The results show that: there are abundant HE existed with immunocytochemical methord and HGCs which secret many zymogen granules with ultrastructure study in the hatching process of embryos of marine shrimp, Penaeus chinensis, in this study. Combine with the result of purification in embryos of marine shrimp, Penaeus chinensis, by using Penaeus chorion as a specific substrate, the HE what we purifying are really the HE of marine shrimp, Penaeus chinensis. As we choose abundant embryos of HE existing, it is undoubtedly think that we successfully get the purification of marine shrimp, Penaeus chinensis.