鲍鱼肌肉质构与呈味变化相关蛋白酶的鉴定、分离与分子克隆

Collagens and myofibrils are major proteins of muscle, contributing to the unique texture of abalone. Matrix metalloproteinases (MMP) are critical proteinases responsible for the metabolism of collagens and myofibrils and the function of aminopeptidase (AP) is relative to the accumulation of free amino acids in abalone muscle. However, no much information is available for the existence of MMP and AP in abalone, including their biochemical properties, primary structures, and the contribution of AP to the taste. In the present study, using abalone (Haliotis discus hannai) muscle as raw material, MMP(s) and AP(s) will be purified to homogeneity by a series of column chromatographies. The biochemical and food chemical characteristics of MMP and AP will be circumspectly investigated. The inner peptide sequences of MMP and AP will also be obtained by MALTI-TOF MS/MS or by protein sequencer analysis. Molecular cloning of MMP and AP will be performed to obtain full length sequences of these proteinases. Based on this research, the enzymatic properties and primary structures of MMP and AP will be elucidated. The effect of MMP on the degradation of abalone collagens, and the effect of AP on free amino acid accumulation, especially their biochemical characteristics will be clarified. Furthermore, the effect of MMP on the textural properties of abalone and the contribution of AP to free amino acid production will also be examined. Combining technologies and methods of protein chemistry, food chemistry, enzymology and biotechnology, the results of the present research will surely be beneficial for adding novel information in basic research on the effective utilization of abalone.

胶原蛋白和肌原纤维蛋白是组成鲍鱼肌肉的主要蛋白，赋予鲍鱼肌肉良好的质构特性。基质金属蛋白酶(MMP)是与胶原蛋白和肌原纤维蛋白新陈代谢密切相关的蛋白酶，而氨肽酶（AP）则与鲍鱼肌肉中游离氨基酸的积累密切相关。但是，鲍鱼肌肉中MMP和AP的存在情况、生化性质、对结构蛋白的分解以及与呈味的相关性等问题仍未有研究报道。本项目拟以我国高产的皱纹盘鲍为研究对象，通过现代生化分离手段从其肌肉中分离纯化MMP和AP，详细分析酶的生物化学特性并利用质谱技术和分子克隆手段获得它们的全序列。通过本研究，将能明确鲍鱼MMP和AP的酶学特性及它们的一级结构，从本质上揭示由MMP引起的鲍鱼肌肉蛋白分解代谢特性与规律，以及对鲍鱼质构特性的影响、AP对游离氨基酸产生的贡献等食品学问题。本研究结合了蛋白质化学、食品化学、酶学和现代生物技术等相关领域的技术手段，研究结果有助于弥补我国在鲍鱼加工利用基础理论研究方面的不足。

本研究以我国高产的皱纹盘鲍为研究对象，利用分子生物学技术对鲍鱼质构变化和呈味相关蛋白酶MMP-1、MMP-2、SP、PEP、组织蛋白酶L和LAP进行分子克隆并确定了它们的一级结构序列。在此基础上，利用荧光定量PCR检测了皱纹盘鲍MMP和LAP表达与季节变化的关系。依据弧菌感染会引起皱纹盘鲍肌肉质构改变的特性，初步探索了MMP和PEP表达和酶活力变化与鲍鱼质构变化的相关性。游离氨基酸对鲍鱼的呈味至关重要，因此检测了AP活性变化与游离氨基酸积累的相关性。此外，弧菌感染显著上调SP和组织蛋白酶L表达水平，而SP通过调控免疫因子表达和细胞吞噬活性来参与鲍鱼抗弧菌免疫反应。.运用原核表达系统获得MMP-1、MMP-2、SP和PEP重组蛋白，其中重组PEP蛋白具有酶活力，结合X-射线衍射技术解明了重组PEP蛋白的晶体结构。利用现代分离手段纯化得到组织蛋白酶L和AP蛋白，详细分析了天然组织蛋白酶L、AP蛋白和重组PEP蛋白的生物化学特性。.在以上研究基础上，还分析了生鲜、煮制、干制、罐制等加工方式以及冷冻时间和温度对鲍鱼模拟胃肠液消化特性和微观结构的影响。从鲍鱼肌肉消化终产物中分离得到一条ACE抑制肽IIMIRF，为降压药的开发提供参考和理论依据。.本研究结合了蛋白质化学、食品化学、酶学和现代生物技术等相关领域的技术手段，研究结果有助于弥补我国在鲍鱼加工利用基础理论研究方面的不足。鲍鱼MMP、SP、PEP、LAP和组织蛋白酶L基因的获得可为今后开发新型酶制剂提供理论依据。