桃多酚氧化酶的纯化及其特性

On Purification and Characterization of Polyphenol Oxidase from Zaohualu Peach

为延长桃果实货架寿命及提高贮藏保鲜技术,以5年生早花露桃果实为试材,对桃多酚氧化酶的纯化及其特性进行了研究,结果表明采用疏水柱层析,透析与PEG浓缩,纯化了早花露桃的多酚氧化酶.所得的酶液经薄层聚丙烯酰胺凝胶等电聚焦电泳,显示单一条带,等电点为3.0.经SDS-PAGE分析,它由2条主要多肽(相对分子质量(MW)分别为3.98×104,6.59×104)和2条含量较少的多肽(MW为6.67×104,6.76×104)所组成.纯化酶液的最高吸收峰在波长272.5nm处,300nm以上几乎无吸收.该酶对邻位二元酚有较高亲和力,几乎不能催化一元酚和三元酚的氧化.以儿茶酚为底物,其最适pH为7.0,最适反应温度20℃.在果实生长发育过程中,多酚氧化酶活性呈下降趋势,中期有一活性相对较小的峰,同时,多酚氧化酶疏水特性增强.

Polyphenol oxidase (PPO) from Zaohualu peach fruits was isolated and purified by aceton powder preparation, Phenyl Sepharose CL-4B hydrophobic chromatography, dialysis and PEG concentration. The purification of 12-fold yielded an apparently homogenous preparation. The isoelectric point is estimated to be 3.0. It reveals two dominant bands with molecular weights of 3.98×104 and 6.59×104 dalton respectively, and two subordinate bands with molecular weights of 6.67×104 and 6.76×104 dalton respectively on SDS-PAGE. The absorption spectrum of the preparation shows maximum at 272.5 nm. There is no significant absorption above 300 nm. The PPO possesses high affinity for O-diphenolic compounds, and catalysed hardly the hydroxylation of monophenol and the oxidation of triphenol. The temparature and pH optima of the enzyme were 20 ℃ and 7.0 respectively. The PPO activity declined during fruit development of Zaohualu peach, and a relatively small activity peack was observed at middle period of the fruit maturity. Meanwhile, the hydrophobic properties of PPO increased and more hydrophobic isoenzymes of PPO appeared as the fruit matured.