基于过渡态底物研究莲子多酚氧化酶促褐变机制

Rich in polyphenols and tender tissue, enzymatic browning catalyzing is easily happened in fresh Lotus Seeds due to the Polyphenol Oxidase (PPO), which seriously affecting the nutritional and economic value of fresh Lotus Seeds. Currently, the enzymatic browning mechanism and efficient inhibitors or preservations during storing and processing of Lotus Seeds becomes key scientific problems to be studied out and solved. The affinity between Transition State Substrate (TSS) and enzyme of the enzyme-catalyzed reaction process is far greater than the ground substrate; it could be more closely combined with the enzyme, and is not easy to change with the substrate concentration. It is essential to determine the TSS to study and clarify the enzymatic browning mechanisms of Lotus Seeds. The Analogs of TSS is suggested to be an efficient inhibitor of the enzymatic browning. In this project, appropriate phenols are selected as target substrates, X-ray diffraction, Circular Dichroism, fluorescence probe and other methods are used, to determine the binding mode during the substrate and Lotus Seeds PPO (LS-PPO). Molecular modeling and Micro-Calorimetry are used to analysis the reaction thermodynamic and kinetic parameters. The possible structure of TSS is speculated using Transition State Theory and Quantum Chemical Theory. The enzymatic browning mechanisms of Lotus Seeds is explored and clarified according to the binding mode and the structure of TSS. TSS is going to be further studied to find out a safe and effective enzymatic browning inhibitor or preservation for storing and processing of Lotus Seeds.

新鲜莲子因酚类物质丰富组织娇嫩极易发生酶促褐变，严重影响其营养及经济价值。研究莲子酶促褐变机制，寻找有效抑制褐变的保鲜剂是当前莲子贮藏加工中亟待解决的关键问题。酶催化反应过程中的过渡态底物对酶的亲和力远远大于基态底物，且与酶的结合更为紧密，其作用不易随底物浓度而改变。过渡态底物的确定有助于理解和阐明莲子PPO促褐变的生化机制。因此，本项目拟从莲子多酚中选择最适莲子PP0作用的靶底物作为最适特异性酚类底物，采用X-衍射、园二色谱及荧光探针等方法确定莲子PPO与最适特异性酚类底物之间的结合方式、结合位点以及结合前后各自构型、构象的变化，再由褐变反应的过渡态底物切入，采用分子模拟、微量热法研究反应动力学及热力学性质，结合过渡态理论及量子化学理论分析过渡态底物变化及其结构特征，尝试基于过渡态底物探讨并阐明莲子PPO促褐变的生化机制，为寻找安全高效的莲子抑制褐变及储藏保鲜方法提供新思路。

新鲜莲子因酚类物质丰富组织娇嫩极易发生酶促褐变，严重影响其营养及经济价值。研究莲子酶促褐变机制，寻找有效抑制褐变的保鲜剂是当前莲子贮藏加工中亟待解决的关键问题。项目通过优化分离与纯化的条件，得到了高纯度的莲子PPO并进行质谱鉴定，确定了莲子PPO的分子量及氨基酸序列等结构信息，构建了莲子PPO的结构模型；分离鉴定并定性定量分析了莲子中的典型酚类底物，结合莲子 PPO 促褐变动力学参数确定了最适特异性酚类底物为表儿茶素；基于莲子PPO的氨基酸序列等结构信息，通过同源建模构建了莲子PPO的三维结构，并通过活性位点预测，发现了莲子PPO的活性中心。通过计算机辅助软件的CDOCKER模块模拟了莲子PPO与特异性底物相互作用的过程，研究了莲子PPO结构对不同底物活性和对映选择性改变的关键位点，准确定位了参与PPO底物识别过程的氨基酸残基，以及相关结构功能域的构象变化，并基于此探讨了莲子 PPO 促褐变的生化机制，为寻找安全高效的莲子抑制褐变及储藏保鲜方法提供新思路。