摘要
为了减少多酚氧化酶(PPO)对产品所带来的负面影响,对从苏北红麦中提取的PPO粗提物进行分离纯化。经硫酸铵沉淀、DEAE阴离子交换层析和Superdex 200凝胶过滤层析,最终得到电泳纯化后只有一条条带的PPO,并对该纯化后的PPO进行酶学性质研究。试验结果为:纯化后PPO总酶活回收率为7.03%,纯化倍数22.35,比酶活为373.44 U/mg,电泳分析表明其相对分子质量约为30 000。测得其最适反应p H为6.5,最适反应温度为37℃;金属离子影响的研究表明,K^+、Na^+对其活性基本无影响,Ca^(2+)、Mg^(2+)、Mn^(2+)略有激活作用,Zn^(2+)、Cu2+激活作用较强;小麦PPO对邻苯二酚的K_m值为6.90 mmol/L,对焦性没食子酸(三酚类)、邻苯二酚(二酚类)底物亲和性较强,对单酚类(苯酚、愈创木酚)、二酚类(对苯二酚、间苯二酚)底物亲和性很低。
In order to minimize the negative impact on products,PPO was purified from the red wheat of north Jiangsu. By ammonium sulfate precipitation,DEAE anion exchange chromatography and Superdex 200 gel filtration chromatography. Finally,only one stripe of PPO was obtained after purified in electrophoresis and the properties of purified PPO were studied. The results were as follows: the enzyme was purified 22. 35 fold with a recovery rate of7. 03% and the specific enzyme activity reached 373. 44 U/mg; Optimum reaction p H was 6. 5,and the optimum reaction temperature was 37 ℃; the effects of metal ions indicated tha the K^+and Na^+had almost no effect on the activity,while Ca^(2+),Mg^(2+)and Mn^(2+)activated the enzyme slightly; the Zn^(2+)and Cu^(2+)stimulated PPO activity obviously; wheat PPO compared to K_mof catechol was 6. 90 mmol/L the PPO had a a strong substrate affinity to pyrogallic acid( three phenols) and catechol( diphenols),but low substrate affinity to bisphenols( phenol,guaiacol),diphenols( hydroquinone,resorcinol).
出处
《中国粮油学报》
EI
CAS
CSCD
北大核心
2016年第5期11-16,共6页
Journal of the Chinese Cereals and Oils Association
关键词
多酚氧化酶
分离纯化
酶学性质
wheat polyphone oxidase
purification
enzymology property