去折叠态多酚氧化酶的酶促褐变与分子构象变化的关系

项目名称： 去折叠态多酚氧化酶的酶促褐变与分子构象变化的关系

项目编号： No.31460435

项目类型： 地区科学基金项目

立项/批准年度： 2015

项目学科： 农业科学

项目作者： 刘伟

作者单位： 南昌大学

项目金额： 55万元

中文摘要： 多酚氧化酶(PPO)的酶促反应在食品贮运加工过程中具有重要的意义。前期发现动态高压微射流处理PPO去折叠态构象变化与酶学特性密切相关以及柠檬酸诱导去折叠态PPO导致其活化能和热力学参数下降，但去折叠态PPO分子构象变化与底物酚类物的柔性机制及不同构象下酶促反应历程的差异性尚不清楚。为此，本项目拟以食品加工过程常见的酸热环境诱导去折叠态PPO为对象，以邻苯二酚为底物，研究其酶促反应（活性、稳定性、反应动力学、热力学、褐变参量）和分子构象变化（活性位点铜离子形态、二级结构、三维构象与氨基酸残基、巯基、二硫键和表面疏水性、折叠动力学、去折叠模型、分子动力学模拟）的影响，比较去折叠态PPO催化邻苯二酚氧化反应历程的差异性，分析构象变化与底物的亲和性，通过蛋白质结构预测和因素变化关系模型建立，揭示去折叠态多酚氧化酶酶促反应与分子构象变化的关系，为进一步理解蛋白质折叠理论和酶促褐变机理提供理论参考。

中文关键词： 多酚氧化酶；去折叠态；分子构象；酶促反应

英文摘要： The enzymatic reaction of polyphenoloxidase (PPO) palys a significant role on the storage and transportation of food. In precious study, it was observed that Dynamic high pressure microfluidization (DHPM) induced unfolding of PPO, which was closely related to the changes of its enzymatic characteristics; and unfolding of PPO induced by citric acid contributed to the decrease of activation energy and thermodynamic parameters of enzyme inactivation. However, the flexible mechanism between the conformational changes of unfloded PPO and phenol substrate and the differences of enzymatic reaction process at the different conformational PPO are still unclear. On the base of those observations, the unfolding of PPO induced by the most common acid and heat treatment is chosen as investigated materials and catechol is used as substrate. Then, enzymatic reaction (activity, stability, reaction kinetics, thermodynamics and browning parameters) and conformational changes (copper ions (in active site) form, secondary structure, three-dimensional structure and amino acid residue, hydrosulphonyl, disulfide bond, surface hydrophobicity, folded kinetics,unfolded model, molecular dynamics simulation ) are studied. The difference of the reaction of unfolding PPO catalyzing catechol is compared. The affinity and the flexibility of conformational changes of unfolded PPO to substrate are analyzed. The correlation between enzymatic reaction and conformational change of unfolded PPO is disclosed by establishing the relationship model between conformational changes and the changes of factors. It is provided a theory guide for further understanding the mechanism of enzymatic browning of PPO.

英文关键词： polyphenoloxidase;unfold;conformation;enzymatic reaction