| 摘要: |
| 采用非变性电泳结合特异性底物发色方法从菲律宾蛤仔血细胞中分离出一种酚氧化酶, 研究了该酶的酶促反应动力学参数, 金属离子、抑制剂对酶活的影响。结果表明, 分离获得的酚氧化酶分子量约为563kDa; 对底物邻苯二酚、左旋多巴(L-DOPA) 、多巴胺、对苯二酚的米氏常数Km分别为0.42 、1.60 、1.79 和1.76mmol/L; 酶的活性在Mg2+、Zn2+、Mn2+、Fe2+、Cu2+和Ca2+浓度为5 、10、20、30mmol/L 时受到抑制; 酶的活性可被抑制剂抗坏血酸、二乙基二硫代氨基甲酸钠(DETC) 、半胱氨酸、亚硫酸钠、柠檬酸、乙二胺四乙酸二钠(EDTA) 、叠氮钠和硫代尿素等抑制。根据底物特异性、抑制剂等结果, 所分离获得的酚氧化酶属于漆酶型的含铜酚氧化酶。 |
| 关键词: 菲律宾蛤仔, 血细胞, 漆酶型酚氧化酶, 金属酶 |
| DOI：10.11693/hyhz201202015015 |
| 分类号: |
| 基金项目:国家自然科学基金资助项目, 30901112 号; 教育部新世纪优秀人才支持计划项目, NCET-10-0763 号; 教育部留学归国人员科研启动金, 教外司留[2011]1139 号。 |
附件 |
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| PURIFICATION AND CHARACTERIZATION OF LACCASE-TYPE PHENOLOXIDASE FROM THE CLAM RUDITAPES PHILIPPINARUM |
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JIANG Jing-Wei, XING Jing, ZHAN Wen-Bin
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The Key Laboratory of Mariculture , Ministry of Education, Ocean University of China
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| Abstract: |
| In this paper, a phenoloxidase (PO) of clam Ruditapes philippinarum was purified from hemocytes using linear-gradient native-PAGE combined with catechol staining. The results showed that this PO had a molecular mass of 563kDa in native-PAGE. Kinetics determination indicated that the Km values of PO for catechol, L-DOPA, dopamine and hydroquinone were 0.42, 1.60, 1.79 and 1.76mmol/L, respectively. The PO activity was inhibited by Mg2+, Zn2+, Mn2+, Fe2+, Cu2+ and Ca2+ at concentrations of 5, 10, 20, 30mmol/L, and also inhibited by inhibitors including ascorbic acid, sodium diethyldithiocarbamate (DETC), cysteine, sodium sulfite, citric acid, ethylenediaminetetraacetic acid disodium (EDTA), sodium azide and thiourea. Based on the substrate specificity and the results of inhibition, it could be concluded that this PO was a laccase-type copper-containing phenoloxidase. |
| Key words: Ruditapes philippinarum, Hemocytes, Laccase-type phenoloxidase, Metalloenzyme |
