红肉桃皮中多酚氧化酶的酶学特性及结构表征  

Property and characterization of polyphenol oxidase in red-fleshed peach skin

作　　者：黄行健[1] 程玉鑫[1] 刘思宇[1] 徐晓云[1] 胡婉峰[1] 潘思轶[1] 

机构地区：[1]华中农业大学食品科学技术学院/环境食品学教育部重点实验室,武汉430070

出　　处：《华中农业大学学报》2016年第3期114-121,共8页Journal of Huazhong Agricultural University

基　　金：国家自然科学基金项目(31401507)

摘　　要：以红肉桃桃皮中多酚氧化酶（polyphenol oxidase,PPO）作为研究对象,采用Tris-HCl缓冲液进行提取并采用硫酸铵盐析法和柱层析法进行纯化,电泳得到单一的条带。根据米氏常数确定桃皮PPO的最适底物为邻苯三酚,采用分光光度计法测定桃皮中多酚氧化酶的活性,其最适温度为45℃、最适p H值为7.0~7.5,PPO在中性p H值环境中以及55℃以下更为稳定。分别应用圆二色谱仪、荧光光谱仪、动态散射光谱仪对PPO的结构进行表征。圆二色谱表明,该PPO二级结构包含α-螺旋、β-折叠、β-转角以及无规卷曲等结构,其含量分别为50.61%、32.47%、10.83%和6.21%。荧光光谱表明,该PPO最大发射波长为352 nm,相对荧光强度为133.9,表明荧光基团处于极性亲水环境中。动态散射光谱显示,该PPO粒径分布集中在255 nm处,表明其在溶液中以聚集形态存在。The polyphenol oxidase in red-fleshed peach skin was extracted in Tris-HC1 buffer and purified by ammonium sulfate precipitation and column chromatography. One pure single band was ob- tained using electrophoresis. According to the Michaelis-Menten constant, the optimal substrate of the PPO was pyrogallol.The optimal temperature and pH of the PPO at 420 nm was 45 ℃ and 7.0-7.5, respectively.PPO was more stable in neutral pH environment and below 55 ℃.Circular dichroism （CD） spectrum,fluorescence spectroscopy, and dynamic light scattering were used to characterize the struc- tures of the PPO.Result of CD spectrum showed that the secondary structure of PPO contained 50.61 α-helix,32.47% β-folded, 10.83% Q-corner and 6.21% random coil. Result of fluorescence spectroscopy showed that the maximum emission wavelength of PPO was 352 nm.The intensity of fluorescence was 133.9,indicating that the fluorophore was in a polar hydrophilic environment. Result of dynamic light scattering showed that the particle size distribution was concentrated at 255 nm,indicating that the PPO was in a gathering state.

关 键 词：红肉桃皮 多酚氧化酶 酶促褐变 酶学性质 结构表征 

分 类 号：TS255.1[轻工技术与工程—农产品加工及贮藏工程]