富士苹果中膜结合态多酚氧化酶分离纯化方法

Isolation and Purification Method of Membrane-bound Polyphenol Oxidase in Fuji Apple

以烟台红富士为原料,研究膜结合态多酚氧化酶（m PPO）的分离纯化条件,采用非离子型去污剂结合热诱导方法粗提,硫酸铵分级沉淀,DEAE Sepharose Fast Flow离子交换柱层析方法对m PPO进行纯化,并对其进行串联质谱（LC-MS/MS）分析鉴定。结果表明m PPO提取纯化最佳条件为：超声提取10 min,50%～80%饱和度硫酸铵沉淀,0.1 mol/L Na Cl梯度洗脱。纯化后的m PPO纯度提高了64.30倍,比活力高达387 032.97 U/mg。纯化后的m PPO经SDS-PAGE和Native-PAGE分析均为单一蛋白带,且为亚基分子量67 k Da的二聚体。经串联质谱分析及蛋白数据库鉴定此纯化蛋白为多酚氧化酶。

Enzymatic browning was mainly associated with polyphenol oxidases which were able to act with phenolic compounds in the presence of molecular oxygen and decreased the commercial quality, organoleptic acceptance and nutritional value of the product. Membrane-bound polyphenol oxidase （mPPO） in Fuji apple （Malus domestica Borkh. cv. Red Fuji） was purified by using temperature- induced phase partitioning technique, ammonium sulfate fractional precipitation and DEAE Sepharose Fast Flow ion exchange column chromatography, and analyzed by using tandem mass spectrometry （LC - MS/MS）. The best conditions for isolation and purification were ultrasonic extraction for 10 min, 50% 80% saturation of ammonium sulfate precipitation, and gradient elution with 0.1 mol/L NaC1. The mPPO was purified by 64. 30 folds with a high specific activity （387 032.97 U/mg）. The Native- PAGE and SDS-PAGE were single bands for mPPO and the purified mPPO was a dimmer of a subunit with a molecular weight of 67 kDa. The results by mass spectrometry analysis and comparison in protein database showed that the purified protein was a polyphenol oxidase.