八棱海棠MdPPO2B基因的表达及功能分析

Expression and Functional Analysis of MdPPO2B from Malus robusta

多酚氧化酶(PPO)是一种含多个铜离子的氧化还原酶,可催化酚类物质氧化为醒类物质。我们从八棱海棠中克隆到一个PPO的同源基因MdPPO2B,构建该基因酵母分泌表达载体并转化到毕赤酵母GS115中。SDS-PAGE电泳结果显示,表达蛋白MdPP02B的分子量约为66.4 kDa。以邻苯二酚作为底物,该酶的最适pH和温度分别为6.5和40℃。除Cu^(2+)、Na^+、Mg^(2+)和Li^+外,4 mmol·L^(-1)的Fe^(3+),Zn^(2+),Mn^(2+)对酶活力表现出较强的抑制作用。在最适条件下,当苯酚浓度达600μmol·L^(-1)时,反应4h,MdPPO2B表达菌株可降解80%的苯酚。表明该基因在工业污水处理中具有广阔的应用潜力。

Polyphenol oxidase（PPO） is one of oxidoreductase enzymes,which catalyzes the phenols to diquinones.A putative PPO gene（MdPP02B） was cloned from Malus robusta and exogenously expressed in Pichia pastoris GS115.SDS-PAGE showed that MdPP02 B has a molecular mass of approximately 66.4 kDa.With catechol as substrate,it was showed that the optimum pH and temperature for MdPP02 B was 6.5 and 40 °C,respectively.Except for Cu^（2＋）,Na~＋,Mg^（2＋） and Li~＋,Fe^（3＋）,Zn^（2＋） and Mn^（2＋） inhibited the enzyme activity at a concentration of 4 mmol·L^（-1）.The MdPPO2 B degraded 80%phenol after incubated for 4 h under the optimum condition.In short,our original research on MdPPO2 B may provide reference for the application of microorganism in industrial wastewater.