Périé F H, Reddy G V, Blackburn N J, Gold M H
Department of Biochemistry and Molecular Biology, Oregon Graduate Institute of Science and Technology, Portland 97291-1000, USA.
Arch Biochem Biophys. 1998 May 15;353(2):349-55. doi: 10.1006/abbi.1998.0625.
Two chromatographic forms of laccase c1 and c2 were purified approximately 225-fold from the extracellular culture fluid of ligninolytic cultures of Dichomitus squalens, using DEAE-Sepharose and Mono-Q fast protein liquid chromatography. Each homogeneous laccase (c1 and c2) has a molecular mass of approximately 66 kDa as determined by SDS-PAGE. Both forms are glycoproteins, and each contains four copper atoms per molecule of protein. The first 20 amino acids of the N-terminal sequences of these two laccases are identical and are similar to those of laccases from other lignin-degrading fungi. The electronic absorption spectra of these laccases exhibit bands at 610 and 330 nm, indicative of type I and type III copper. The EPR spectrum of laccase c1 exhibits bands indicative of type I and type II copper. Each laccase oxidizes a variety of phenolic substrates, has a pH optimum of 3.0 for the oxidation of 2,6-dimethoxyphenol, and is inhibited strongly by fluoride and azide.
使用DEAE-琼脂糖和Mono-Q快速蛋白质液相色谱法，从黄孢原毛平革菌木质素分解培养物的细胞外培养液中，将漆酶c1和c2的两种色谱形式纯化了约225倍。通过SDS-PAGE测定，每种均一的漆酶（c1和c2）的分子量约为66 kDa。两种形式均为糖蛋白，且每分子蛋白质均含有四个铜原子。这两种漆酶N端序列的前20个氨基酸相同，且与其他木质素降解真菌的漆酶相似。这些漆酶的电子吸收光谱在610和330 nm处有吸收带，表明存在I型和III型铜。漆酶c1的EPR光谱显示出表明存在I型和II型铜的吸收带。每种漆酶均可氧化多种酚类底物，氧化2,6-二甲氧基苯酚的最适pH为3.0，且强烈受氟化物和叠氮化物抑制。
