云芝漆酶的分离纯化和理化性质及抗增殖活性

Purification and Characterization of a Laccase with Antiproliferative Activity from Coriolus versicolor

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。

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.