芋头多酚氧化酶的分离纯化与酶学特性  

Separation,Purification and Enzymatic Characterization of Polyphenol Oxidase from Taro

作　　者：崔晓颖 彭新颜 贺红军[1] 张敏[1] 张晓彤 CUI Xiaoying;PENG Xinyan;HE Hongjun;ZHANG Min;ZHANG Xiaotong(College of Life Sciences,Yantai University,Yantai 264005,China;Bio-Nanotechnology Institute,Ludong University,Yantai 264025,China)

机构地区：[1]烟台大学生命科学学院,山东烟台264005 [2]鲁东大学生物纳米技术研究院,山东烟台264025

出　　处：《食品科学》2021年第12期107-115,共9页Food Science

基　　金：国家自然科学基金面上项目(32072141);山东省重点研发计划公益类项目(2019GSF108011)。

摘　　要：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。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 27471.26 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.

关 键 词：芋头 酶促褐变 多酚氧化酶 分离纯化 酶学特性 

分 类 号：TS255.3[轻工技术与工程—农产品加工及贮藏工程]