苜蓿二胺氧化酶(DAO)的分离纯化

Separation and Purification of Diamine Oxidase from Alfalfa

doi:
10.3969/j.issn.0517-6611.2017.28.048
摘要:
[目的]寻求操作简单、高效的苜蓿二胺氧化酶(DAO)纯化方法.[方法]苜蓿种子黑暗培养3 d,幼苗依次经硫酸铵沉淀,葡聚糖凝胶G-100(sephadex G-100),DEAE-纤维素以及羟基磷灰石(hydroxyapatite,HAP)层析分离纯化,最后经聚丙烯酰胺凝胶电泳.[结果]最佳分段盐析的硫酸铵饱和度为20%~50%.经一系列层析分离后酶液比活力达84.67 U/mg,回收率为26%.经非变性聚丙烯酰氨凝胶电泳后,出现1个蛋白质条带,相对分子质量约为110 kDa.而在变性聚丙烯酰氨凝胶电泳后,出现2个蛋白质条带,相对分子质量约为55和40 kDa.[结论]苜蓿DAO是由2个大小不同的亚基组成的异源二聚体.
Abstract:
[Objective]To seek a simple and efficient method for purifying the diamine oxidase from alfalfa.[Method] Alfalfa seeds were cul-tured in darkness for 3 days.The seedlings were isolated and purified by ammonium sulfate precipitation, dextran gel G-100 ( sephadex G-100), DEAE-cellulose and hydroxyapatite (HAP),finally, polyacrylamide gel electrophoresis.[Result] The best fraction of salting out am-monium sulfate saturation was 20% -50%.After a series of chromatographic separation, the specific activity of the enzyme reached 84.67 U/mg, and the recovery rate was 26%.After the non-denaturing polyacrylamide gel electrophoresis, a protein band appeared with a relative molecular mass of about 110 kDa.But after denaturing polyacrylamide gel electrophoresis, two protein bands appeared at a relative mo-lecular mass of about 55 kDa and 40 kDa.[ Conclusion] Diamine oxidase from alfalfa is a heterodimer composed of two subunits of different si-zes.
作者 穆文静    张永明    红格日其其格
Author: MU Wen-jing    ZHANG Yong-ming    HONG Geri-qiqige
作者单位 内蒙古师范大学生命科学与技术学院,内蒙古呼和浩特,010022
刊 名: 安徽农业科学   ISTICPKU
Journal: Journal of Anhui Agricultural Sciences
年,卷(期) 2017, 45(28)
分类号 S188
关键词: 苜蓿 知识脉络    二胺氧化酶 知识脉络    分离纯化 知识脉络   
Keywords: Alfalfa    Diamine oxidase    Separation and purification   
机标分类号
基金项目 国家自然科学基金项目,内蒙古教育厅项目
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