芋头多酚氧化酶的分离纯化与酶学特性

以鲜芋头为原料，通过提取、30%～80%饱和度硫酸铵沉淀、透析、超滤，以及柱层析等步骤对芋头多酚氧化酶（polyphenol oxidase，PPO）进行逐级分离纯化，并探讨其酶学特性及抑制剂对其作用规律。结果表明：经DEAE-Sepharose、Superdex G-75色谱柱纯化后，得到了电泳纯PPO，比活力为27 471.26 U/mg，纯化倍数达6.37 倍。纯化后的PPO经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳分析，其分子质量约为24 kDa。PPO与底物结合能力顺序为：邻苯二酚＞间苯二酚＞间苯三酚＞焦性没食子酸＞L-酪氨酸。PPO的最适反应温度、pH值和反应时间分别为30 ℃、6.8和60 s。抗坏血酸和L-半胱氨酸对提取的PPO有较强的抑制作用，其抑制类型分别属于反竞争性抑制和竞争性抑制方式。结果表明，在芋头加工过程中通过控制温度、pH值或加入适当的抑制剂处理，可以有效减少褐变的发生。

Separation,Purification and Enzymatic Characterization of Polyphenol Oxidase from Taro

Polyphenol oxidase (PPO) from taro was obtained and purified through sequential steps of extraction, precipitation with 30%–80% saturated ammonium sulfate, dialysis, ultrafiltration, and chromatographic separation. The PPO was enzymatically characterized and the action pattern of selected inhibitors on it was explored. The results showed that electrophoretically pure PPO was obtained using DEAE-Sepharose and Superdex G-75 column chromatography, and the specific activity was 27 471.26 U/mg, which corresponded to a 6.37-fold purification. SDS-PAGE analysis of the purified PPO showed one band at approximately 24 kDa. The enzyme showed high substrate affinity for catechol. Its binding capacity to various substrates decreased as follows: catechol > resorcinol > phloroglucinol > pyrogallic acid > L-tyrosine. The optimal reaction temperature, pH and time were 30 ℃, 6.8 and 60 s, respectively. The PPO enzyme was inhibited by ascorbic acid and L-cysteine in an uncompetitive and competitive manner, respectively. This study demonstrates that controlling the temperature and pH and adding inhibitors can reduce the occurrence of enzymatic browning during taro processing effectively.