【作者】马茜茜; 李淼; 王贺祥
【Author】MA Qianqian; LI Miao; WANG HexiangState Key Laboratory for Agrobiotechnology and Department of Microbiology; China Agricultural University; Beijing 100193; China
【作者机构】中国农业大学生物学院农业生物技术国家重点实验室
【摘要】 采用DEAE-cellulose,CM-cellulose和Q-Sepharose离子交换层析和Superdex 75凝胶过滤层析,从云芝(Coriolus versicolor)发酵液中纯化得到了一种漆酶,SDS-PAGE电泳检测其为单一蛋白,分子量为67 kDa,N-端部分氨基酸序列为GIGPVADLTITNAAV。该酶可以催化氧化多种底物,其中对ABTS[2,2,-azinobis-(3-ethylbenzothiazoline-6-sulphonate)]的催化作用最强。最适反应条件为40℃,pH 5.0,其活性可以被Cu2+激活,被Fe3+、Hg2+、Fe2+抑制。该酶对HIV-1逆转录酶、乳腺癌MCF7细胞和肝癌HepG2细胞均具有抑制作用,其IC50分别为1.5μmol/L、2.3μmol/L和4.4μmol/L。
【Abstract】 A laccase from Coriolus versicolor was purified by ion exchange chromatography(DEAE-cellulose,CM-cellulose,Q-Sepharose) and gel filtration(Superdex 75) using fast protein liquid chromatography(FPLC).The enzyme is a monomeric protein with a molecular mass of 67 kDa based on SDS-PAGE and an N-terminal amino acid sequence of GIGPVADLTITNAAV.Purified laccase oxidized a range of phenolic and non-phenolic substrates,and exhibited highest activity on 2,2-azinobis-(3-ethylbenzothiazoline-6-sulphonate)(ABTS).With ABTS as the substrate,the enzyme displayed pH and temperature optima of 5.0 and 40 ℃,respectively and was significantly inhibited by Fe3+、Hg2+、and Fe2+ but stimulated by Cu2+.C.versicolor laccase inhibited HIV-1 reverse transcriptase activity and the proliferation of MCF7 and HepG2 cancer cells with IC50 values of 1.5 μmol/L,2.3 μmol/L and 4.4 μmol/L,respectively.
【关键词】 云芝; 漆酶; 分离纯化
【Key words】 Coriolus versicolor; laccase; enzyme purification
【基金】科技部“十一五”支撑项目“京郊地区种养业废弃资源循环利用技术集成研究与示范”(编号:2007BAD89B07);; 中国农业大学生物学理科基地能力提高项目(科研训练,J0730639)的部分研究内容~~
【文献出处】 食用菌学报,Acta Edulis Fungi,编辑部邮箱,2010年02期
【DOI】CNKI:SUN:SYJB.0.2010-02-009
【分类号】S567.31