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

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pH值为7.0~7.5，PPO在中性pH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-HCl buffer and purified by ammonium sulfate precipitation and column chromatography.One pure single band was obtained 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 structures 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% β-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.