香豆素衍生物对梨汁多酚氧化酶的抑制机理

The study of inhibition mechanisms on polyphenol oxidase (PPO) during the fruit juice browning is one of the frontier problems in food sciences. However, The inhibiting mechanisms of coumarin derivatives on PPO is not clear. Thus, In this project, we selected the coumarin derivatives as subjects. We extracted the PPO from pear juice, studied the characteristics of PPO, analyzed the kinetics after treated with the coumarin derivatives, and obtained as the inhibition of PPO in coumarin derivatives. Using the gel electrophoresis, we analyzed the stripe number of PPO in the primary structure. Using the fourier transform infrared spectroscopy and circular dichroism, we analyzed the α-helix, β-sheet, β-turn and random coil of PPO in the secondary structure. Using fluorescence spectroscopy and nuclear magnetic resonance, we analyzed the structural domain of PPO in the tertiary structure. Using the transmission electron microscopy, we analyzed the form of PPO in the quaternary structure. The regulating characteristics of coumarin derivatives on the structure of PPO were revealed. On the base of known crystal structures of PPO, we built the molecular docking model, obtained the action modes and binding sites between the coumarin derivatives and PPO, and revealed the functional relations between the types of substituent and location of coumarin derivatives and PPO. These results of this project illuminate the inhibition mechanism of coumarin derivatives on PPO in pear juice, providing the important theories and values for enzymatic browning of juice and synthesis of coumarin derivatives.

果汁褐变过程中多酚氧化酶（PPO）抑制机理的研究是当今食品科学的前沿问题，但香豆素衍生物对PPO的抑制机理尚未明晰，故本项目拟以香豆素衍生物作为研究对象，提取梨汁中PPO，研究其酶学特性，分析香豆素衍生物处理PPO后动力学变化，得到具有抑制作用的香豆素衍生物；采用电泳分析PPO一级结构中蛋白条带数目变化，采用傅立叶变换红外光谱和圆二色谱分析二级结构中α-螺旋、β-折叠、β-转角及无规则卷曲变化，采用荧光光谱和核磁共振分析三级结构的结构域变化，采用透射电子显微镜分析四级结构的形态变化，揭示香豆素衍生物处理前后PPO结构变化规律；在已知PPO晶体基础上建立分子对接模型，获得香豆素衍生物与PPO的作用方式和结合位点，揭示不同取代类型和位置的香豆素衍生物与PPO钝化之间的关联性。研究结果将阐明香豆素衍生物对梨汁PPO的抑制机理，在果汁酶促褐变以及香豆素衍生物合成等方面具有重要理论意义和应用价值。

本实验选取了25种香豆素类化合物，以L-多巴为底物测定它们对多酚氧化酶（PPO）的抑制作用，探讨不同类型的香豆素类化合物对PPO的抑制作用的构效关系。从25种香豆素类化合物中筛选出了对PPO抑制效果最强的4-羟基香豆素，其半数抑制浓度为0.37 mmol•L-1，当4-羟基香豆素浓度为3.33 mmol•L-1时，PPO剩余酶活力仅5%，因此选择4-羟基香豆素作为目标抑制剂进行抑制机理研究。. 通过酶抑制动力学判断它对PPO的抑制类型为可逆竞争型抑制，并得到米氏常数为1.685 mmol•L-1；凝胶电泳结果显示4-羟基香豆素作用前后PPO蛋白条带数目与位置均未发生变化，即4-羟基香豆素对PPO一级结构无影响。圆二色光谱结果显示4-羟基香豆素作用后酶蛋白α-螺旋含量显著降低，表明PPO二级结构发生了变化，酶表面的疏水作用增强。同时PPO内源荧光光谱最大发射峰蓝移，说明色氨酸残基的微环境极性降低，且透射电镜结果显示处理后的PPO发生聚集，直径增大，均印证了处理后的酶表面的疏水作用增强，导致酶之间由于疏水作用力的增强而聚集，以致底物难以与酶的结合位点相结合，从而抑制酶的活性。. 分别选取不同品种的西洋梨和亚洲梨进行应用试验，4-羟基香豆素浓度为2 mmol•L-1均取得良好的抑制效果。