海参体壁自溶关键蛋白酶及其作用机理研究

Serine proteinases (SP) and matrix metalloproteinases (MMP) are critical proteinases responsible for the autolysis of collagens. The effect of these two kinds of proteinases are major reasons for sea cucumber body wall degradation and decomposition. Though previous studies reported the existence of cysteine proteinase(s) in sea cucumber body wall, till now, no much information is available for biochemical properties, primary protein structures, processing mechanism and especially food chemical properties of these proteinases. In the present study, using sea cucumber (Stichopus japonicus) as raw material, critical serine proteinase(s) and matrix metalloproteinase(s) will be purified to homogeneity by a series of column chromatographies. The biochemical and food chemical characteristics of SP and MMP will be circumspectly investigated. The inner peptide and N-terminal sequences of SP and MMP will also be analyzed by MALTI-TOF mass spectrum and protein sequencer analysis. After extration of total RNA from body wall and synthesis of the first strand cDNA, molecular cloning of SP and MMP will be performed to obtain full length sequences of these proteinases. Based on this research, the enzymatic properties and primary structures of SP and MMP from sea cucumber will be elucidated. The effect of SP and MMP on the degradation of sea cucumber collagens and especially their biochemical characteristics will be disclosed. Furthermore, the effect of SP and MMP on the textural properties of sea cucumber will also be exhibited. Combining technologies and methods in protein chemistry, food chemistry, enzymology and biotechnology, the results of the present research project will surely be beneficial to add new information in the effective utilization of marine bioresources and may establish a firm base for the development of novel proteinases.

丝氨酸蛋白酶(SP)和基质金属蛋白酶(MMP)是与胶原蛋白新陈代谢密切相关的蛋白酶，这两种酶对胶原蛋白的分解作用是导致海参体壁自溶的主要原因。但是，海参体壁自溶酶的生物化学性质、一级结构、作用机理，尤其是酶对胶原蛋白的分解作用等食品学性质仍不明确。本项目拟以刺参为研究对象，通过现代生化分离手段从其体壁中分离纯化关键蛋白酶SP和MMP，详细分析酶的生物化学和食品学特性并利用质谱技术和分子克隆手段获得其全序列。通过本研究，将能明确刺参SP和MMP的酶学特性及它们的一级结构并从原理上揭示由SP和MMP引起的海参胶原蛋白分解代谢等生化特性与规律，SP和MMP对海参体壁质构特性的影响机理等食品学问题也将得到解明。本研究充分结合了蛋白质化学、食品化学、酶学和现代生物技术等相关领域的技术手段和实验方法，研究结果有助于弥补我国在海洋生物资源高效利用基础理论研究方面的不足，也可为开发新型酶制剂提供理论依据。

本研究以具有重要经济价值的刺参(Stichopus japonicas)为研究对象，从其内脏和体壁中分别纯化了丝氨酸蛋白酶（Serine proteinase, SP）和金属蛋白酶（Metalloproteinase, MP），并对它们的生物化学性质及食品学性质进行了深入研究。SP的分子量为34 kDa,最适pH为6.0~9.0，在35~40℃之间表现出较高的胶原蛋白分解活性; MP的分子量为45 kDa，最适pH为8.0~9.0，在40~45 °C之间表现出较高的胶原蛋白分解活性。质谱分析进一步确认了纯化的SP和MP分别为丝氨酸蛋白酶和金属蛋白酶。SP和MP不仅在37°C，甚至在4°C都可以有效降解海参胶原蛋白，因此确认，二者是参与海参自溶过程中降解胶原蛋白的重要蛋白酶。同时，利用分子生物学技术对SP以及基质金属蛋白酶MMP-2进行分子克隆确定它们的一级结构序列。运用原核表达系统获得重组rSP和rMMP-2的体外表达，制备了抗SP多克隆抗体。本研究还对体壁和内脏的SP和MP进行了分离纯化，均表明也可以有效降解海参胶原蛋白。此外，本研究首次克隆获得海参胶原蛋白COLα的cDNA全长序列。该基因长度为4623 bp，开放阅读框4203 bp，共编码1400个氨基酸残基，预测其分子量为131.7 kDa，理论等电点6.17。通过本项目的研究，明确了刺参SP和MP的酶学特性及它们的一级结构并从原理上揭示由SP和MP引起的海参胶原蛋白分解代谢等生化特性与规律。本研究充分利用了蛋白质化学、食品化学、酶学和免疫学等相关领域的技术手段和实验方法，研究结果弥补了我国在重要海洋生物资源海参高效利用基础理论研究方面的不足。海参SP和MP基因的获得可为今后开发新型酶制剂提供理论依据。