马铃薯两种形态多酚氧化酶分离纯化及其性质研究

The Polyphenol Oxidase mediated browning seriously reduce the quality of products processed from potato. Common browning inhibitors do not figure out the enzyme browning totally, because the existing of membrane-bound PPO (mPPO). Therefore, at the present study, we take Solanum tuberosum Linn and Solanum tuberosum Red Emerald as the experimental materials. Firstly, the suitable purification protocol of membrane-bound and soluble PPO(sPPO) are established based on the ammonium sulfate precipitation and anion exchange chromatography column method optimization. Thereafter, the gained mPPO and sPPO are identified through Bio-mass Spectrometry. Secondly, the difference between potato cultivars and these two type of enzymes are measured. Thirdly, following the multiple sequence alignment, structure prediction, molecular docking and chemical modification, the structure , functional groups and browning mechanism of mPPO and sPPO are analysised. Finally, the effect of inhibitors on action center and structure change of mPPO and sPPO are analyzed through their change of ultraviolet absorption spectrum and fluorescence emission spectrum, then the inhibition mechanism of the inhibitors are investigated. Results would contribute to the control of browning in the potato processing.

多酚氧化酶（Polyphenol Oxidase，PPO）引起的酶促褐变严重影响马铃薯加工产品的质量。普通的褐变抑制剂很难从根本上解决酶促褐变问题，这与膜结合态PPO密切相关。因此，本项目拟以黑美人与红宝石马铃薯为实验材料，以硫酸铵沉淀法结合阴离子交换层析柱法，建立适合膜结合态与可溶态多酚氧化酶纯化体系，并进行生物质谱鉴定；然后比较品种间两种形态多酚氧化酶的酶学特性差异；通过多重序列比对、结构预测、分子对接及化学修饰剂修饰，对两种形态多酚氧化酶的结构、必须功能基团及褐变机理进行研究；最后，通过分析膜结合态与可溶态多酚氧化酶紫外吸收光谱及荧光发射光谱变化，推测抑制剂对其活性中心及结构变化的影响，从而揭示抑制剂对不同形态马铃薯PPO的抑制机理。研究结果将为马铃薯加工过程中褐变的控制提供重要的参考依据。

酶促褐变能严重影响马铃薯的生产加工，对制品的色泽、风味、硬度等感官特性造成影响，同时其产生的类黑素聚合体不利于人体消化吸收，使产品的营养价值降低，甚至有可能影响食品的安全性。贵州马铃薯种植面积一直位居全国前三位，但对马铃薯的加工利用较其他省份落后。本课题组为了改变贵州省马铃薯加工现状，已经研制出了薯片、马铃薯净菜、马铃薯面条、米粉、馒头等一系列制品，但在加工过程中发现马铃薯褐变会造成原料的极大浪费，成本增加。因此本研究以薯片加工专用品种大西洋马铃薯、净菜加工品种费乌瑞它马铃薯以及特色马铃薯黑美人和红宝石为研究材料，对马铃薯膜结合态多酚氧化酶（mPPO）与可溶态多酚氧化酶（sPPO）的提取工艺进行响应面优化，并进一步对sPPO和mPPO进行纯化、鉴定，建立了mPPO和sPPO分离纯化技术体系。比较分离到的mPPO和sPPO以及不同品种间两种形态多酚氧化酶的酶学特性差异，确定其活性中心必需基团组成，研究抑制剂对mPPO与sPPO抑制效果及光谱特性的影响，初步揭示抑制剂对mPPO和sPPO的抑制机理。本项目研究结果将为马铃薯加工过程中褐变的控制提供重要的参考依据。