去折叠态多酚氧化酶的酶促褐变与分子构象变化的关系

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.

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。

多酚氧化酶（PPO）主导的酶促褐变导致果蔬营养品质劣变及经济价值下降。目前PPO的研究主要集中于酶学特性及活性抑制方面，关于PPO钝化机理、酶促褐变与分子构象变化关系的研究还很少。因此，本项目通过测定食品加工过程中PPO活性、稳定性、反应动力学及热力学以表征其酶促反应特性，并通过多种光谱学、电子显微镜及分子动力学模拟技术分析PPO分子构象及微观结构变化，从而研究加工过程中诱发的去折叠态PPO分子构象变化和酶促褐变关系。（1）有机酸（肉桂酸、柠檬酸、苹果酸）对PPO不同的抑制机理：肉桂酸为PPO的混合型可逆抑制剂，通过与PPO活性中心附近的组氨酸形成π–π堆积嵌入到PPO的疏水空腔并使PPO二级结构发生重排；苹果酸和柠檬酸作为酸化剂使体系pH的降低是其起抑制作用的主要原因。（2）不同热处理影响PPO分子结构并导致其不同程度失活：高温条件下PPO二级结构发生重排，三级结构被破坏，PPO失活率与其内源荧光光谱红移呈正相关性；中温条件下PPO分子发生变形而高温下易形成聚集态，相同活性PPO可以多种形态存在。（3）酸性条件诱导PPO去折叠构象变化可增强物理方法对PPO钝化效果：弱酸性条件下PPO构象逐渐展开，具有重折叠性，但pH≤4.0时PPO发生严重失活及变性且不可逆，有机酸结合热或超声波处理对PPO的钝化效果明显强于单独使用效果之和。（4）有机酸结合高压或热加工能更好地抑制果蔬产品褐变和保持果蔬品质：柠檬酸结合高压处理有效钝化梨泥PPO活性，提高其总酚含量和氧自由基吸收能力；热处理联合肉桂酸能有效抑制鲜切山药褐变。本项目揭示有机酸、热处理诱发去折叠态PPO活性抑制与分子构象变化密切相关，为果蔬酶促褐变控制提供理论借鉴。本项目圆满完成预期目标，在基金资助期间发表了9篇论文 (SCI论文7篇)，其中在食品TOP期刊Food Chemistry（2017 IF＝4.946）发表论文4篇。课题研究期间培养博士研究生1名、硕士研究生3名。