UV-C胁迫诱导虾头自降解关键酶自活化的分子机制及其肽释放行为的定向调控

Marine animals are easily subjected to self degradation after dead.The proteins in their body tissues are hydrolyzed by the endogenous protoelytic enzymes,the main products are peptides and free amino acids.The procedure are rather complex and it is difficult to control the resulted products.Our research team found in several previous researches that UV-C irradiation stress could induce the self degradation of shrimp tissues. However, the related mechasim is still unclear. The project is going to activate the endogenous enzymes shrimp head of Penaeus vannamei UV-C irradiation stress, build a simulated digestion to prepare short peptides which can be easily absorbed. It will greatly enhance the utilization value of shrimp head waste. The change rules of enzymology properties, molecular conformation, self degradation kineitcs of the endogenous enzymes, physicochemical properties of the substrate protein in the shrimp head before and after UV-C irradiation stress are investigated, the purpose is to elucidate the catalitic mechasim of the self degradation of shrimp head by UV-C irradiation stress; Simulated gastrointestinal digestion system is founed based on enzyme properties and specific inhibitors of the endougenous enzyems, the system combined with kinetic models for controlled enzymatic hydrolysis is used to regulate the peptide release behavior directionaly during the self degradation of shrimp head, short peptides is then prepared; Artificial gastrointestinal fluids and Caco-2 cell model are employed to estimate the esistant capacity to gastrointestinal digestion and the absorptive capacity.In clusion, the present project will illustrate the key proteolytic enzymes caused the self degradation of shrimp head, and reveal activation mechasim of endougenous enzymes by UV-C irradiation stress. The results of the project will provide a new approach for preparing easily absorbed bioactive peptides from food proteins and utilizing the marine biological resources effectively.

海洋水产动物死后易发生自降解作用，其组织蛋白在内源蛋白酶作用下以肽、氨基酸等形式释放出来，整个过程缓慢且产物难以控制。本课题组的前期研究发现适度UV-C辐射胁迫能诱导虾组织自降解，但有关机理尚不明确。本项目拟利用UV-C胁迫诱导凡纳滨对虾虾头内源酶活化，构建模拟消化体系，制备具有良好吸收性能的短肽，以提高其利用价值：通过探究UV-C胁迫前后内源蛋白酶的酶学特性、分子构象、动力学及底物蛋白理化性质的变化规律，阐明紫外胁迫诱导虾头蛋白自降解的机理；通过酶学特性、专一性抑制剂构建模拟消化体系，结合可控酶解模型定向调控虾头自降解过程的肽释放行为，制备短肽产物；通过人工胃肠液、Caco-2模型对自降解短肽进行消化及吸收性能评价。本项目将揭示虾头自降解的关键蛋白酶，阐明UV-C胁迫下内源酶的自活化机制，研究结果将为易吸收型食品蛋白源活性肽的制备及海洋生物资源的高效利用提供新思路。

海洋水产动物死后易发生自降解作用，其组织蛋白在内源蛋白酶作用下以肽、氨基酸等形式释放出来，整个过程缓慢且产物难以控制。本课题组的前期研究发现适度UV-C辐射胁迫能诱导虾组织自降解，但有关机理尚不明确。本项目拟利用UV-C胁迫诱导凡纳滨对虾虾头内源酶活化，构建模拟消化体系，制备具有良好吸收性能的短肽，以提高其利用价值：通过探究UV-C胁迫前后内源蛋白酶的酶学特性、分子构象、动力学及底物蛋白理化性质的变化规律，阐明紫外胁迫诱导虾头蛋白自降解的机理；通过酶学特性、专一性抑制剂构建模拟消化体系，结合可控酶解模型定向调控虾头自降解过程的肽释放行为，制备短肽产物；通过人工胃肠液、Caco-2模型对自降解短肽进行消化及吸收性能评价。本项目经过4年的研究，获得了几个创新性的发现：1）UV-C照射胁迫可以激活虾头内多种内源酶，除内源蛋白酶外，脂酶、多酚氧化酶、几丁质酶均可显著地激活；2）基本阐明了UV-C对虾头内源蛋白酶的激活分子机制：通过UV-C的胁迫，酶蛋白质分子的空间构象发生变化，从而使得其更易与底物蛋白结合发生催化作用，目前的实验结果显示UV-C胁迫不能明显激活酶原；3）可以利用虾头的内源实现模拟消化式的自降解，将自身蛋白最大程度降解成易吸收的低分子肽释放出来。本项目的研究结果在一定程度上阐释了虾头的自降解现象及UV-C对其自降解的促进作用，也为实现虾头的可调控自降解提供了实验依据，为虾头副产物的高值化利用提供了重要的参考。将来在实验参数扩大、研究对象拓展后，对其它水产品的加工副产物蛋白质的回收利用亦有重大参考价值，并且可在相应的技术工艺研发成熟后进行生产应用。