《植物生理学报》 2015, 51(12): 2239-2246

八棱海棠MdPPO2B基因的表达及功能分析

沈雪芳^1,2, 韩晴², 高建杰³, 彭日荷³, 王波³, 高志红¹, 姚泉洪^1,3,*, 章镇^1,*

¹南京农业大学园艺学院, 南京210095; ²上海市农业科学院作物育种栽培研究所, 上海201403; ³上海市农业科学院生物技术研究所, 上海201106

通信作者：姚泉洪；E-mail: yaoquanhong88@163.com, zhangzh@njau.edu.cn；Tel: 025-84395724, 021-62203180

摘　要：

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醌类物质。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MdPPO2B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Cu²⁺、Na⁺、Mg²⁺和Li⁺外, 4 mmol•L^-1的Fe³⁺、Zn²⁺、Mn²⁺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600 μ mol•L^-1时, 反应4 h, 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。

关键词：多酚氧化酶; 八棱海棠; 酶学性质; 苯酚降解

收稿：2015-09-10   修定：2015-10-30

资助：上海市种业发展项目[沪农科种字(2013)第13号]、上海市科学技术委员会科研计划项目(13395800300)。

Expression and Functional Analysis of MdPPO2B from Malus robusta

SHEN Xue-Fang^1,2, HAN Qing², GAO Jian-Jie³, PENG Ri-He³, WANG Bo³, GAO Zhi-Hong¹, YAO Quan-Hong^1,3,*, ZHANG Zhen^1,*

¹College of Horticulture, Nanjing Agricultural University, Nanjing 210095, China; ²Crop Breeding and Cultivation Research Institute, Shanghai Academy of Agricultural Sciences, Shanghai 201403, China; ³Agro-Biotechnology Research Center, Shanghai Academy of Agricultural Sciences, Shanghai 201106, China

Corresponding author: YAO Quan-Hong; E-mail: yaoquanhong88@163.com, zhangzh@njau.edu.cn; Tel: 025-84395724, 021-62203180

Abstract:

Polyphenol oxidase (PPO) is one of oxidoreductase enzymes, which catalyzes the phenols to diquinones. A putative PPO gene (MdPPO2B) was cloned from Malus robusta and exogenously expressed in Pichia pastoris GS115. SDS-PAGE showed that MdPPO2B has a molecular mass of approximately 66.4 kDa. With catechol as substrate, it was showed that the optimum pH and temperature for MdPPO2B was 6.5 and 40 ℃, respectively. Except for Cu²⁺, Na⁺, Mg²⁺ and Li⁺, Fe³⁺, Zn²⁺ and Mn²⁺ inhibited the enzyme activity at a concentration of 4 mmol•L^-1. The MdPPO2B degraded 80% phenol after incubated for 4 h under the optimum condition. In short, our original research on MdPPO2B may provide reference for the application of microorganism in industrial wastewater.

Key words: polyphenol oxidase; Malus robusta; enzymatic characteristics; phenol degradation