Salas C, Lobos S, Larraín J, Salas L, Cullen D, Vicuña R
Laboratorio de Bioquímica, Facultad de Ciencias Biológicas, Pontificia Universidad Católica de Chile, Santiago.
Biotechnol Appl Biochem. 1995 Jun;21(3):323-33.
Laccase is one of the ligninolytic enzymes found in liquid cultures of the fungus Ceriporiopsis subvermispora in defined medium. As an approach to a clarification of the role of laccases during the attack on lignin by the fungus, the enzyme has been characterized further. The levels of this phenol oxidase increase 2-fold in the presence of p-anisidine and are severely affected when addition of either Mn(II) or Cu(II) ions to the medium is omitted. Isoelectrofocusing allowed the resolution of two laccase isoenzymes, with pIs of 3.65 and 3.59. In rich medium, laccase activity is 10-fold higher than in salt medium, and it is not affected by the external addition of p-anisidine or Mn(II). Four isoenzymes were detected in these cultures, with pIs between 3.76 and 3.60. In a wheat bran medium, four isoenzymes with pIs in the range 3.63-3.46, plus a fifth isoenzyme of high pI (4.82), were also identified. The absorption spectrum of a pool containing the four isoenzymes from rich medium shows a maximum at 600 nm, typical of laccase possessing a type I copper atom. The molecular mass of the isoenzyme with pI 3.60 is 79 kDa, as determined by SDS/PAGE. Upon treatment with endoglycosidase F, the molecular mass of this isoform decreases to 63 kDa, indicating a high degree of glycosylation. Substrate specificity studies conducted with the four isoenzymes from rich medium and a combination of isoenzymes from salt medium showed marked differences among them. The amino-terminal sequences (24 residues) of three isoenzymes isolated from rich medium were determined. Two of them are identical, whereas the third one differs from these in three amino acid residues. The consensus sequence reveals clear homology with laccases from other microorganisms.
漆酶是在特定培养基中真菌黄孢原毛平革菌液体培养物中发现的木质素分解酶之一。作为阐明漆酶在真菌攻击木质素过程中作用的一种方法，对该酶进行了进一步的特性分析。在对甲氧基苯胺存在的情况下，这种酚氧化酶的水平增加了2倍，并且当培养基中省略Mn(II)或Cu(II)离子的添加时，其受到严重影响。等电聚焦可分辨出两种漆酶同工酶，其等电点分别为3.65和3.59。在丰富培养基中，漆酶活性比在盐培养基中高10倍，并且不受对甲氧基苯胺或Mn(II)的外部添加的影响。在这些培养物中检测到四种同工酶，其等电点在3.76至3.60之间。在麦麸培养基中，还鉴定出四种等电点在3.63 - 3.46范围内的同工酶，以及第五种高pI（4.82）的同工酶。来自丰富培养基的包含四种同工酶的酶液的吸收光谱在600 nm处显示出最大值，这是具有I型铜原子的漆酶的典型特征。通过SDS/PAGE测定，等电点为3.60的同工酶的分子量为79 kDa。用内切糖苷酶F处理后，该同工型的分子量降至63 kDa，表明其高度糖基化。用来自丰富培养基的四种同工酶和来自盐培养基的同工酶组合进行的底物特异性研究表明它们之间存在明显差异。测定了从丰富培养基中分离出的三种同工酶的氨基末端序列（24个残基）。其中两种相同，而第三种在三个氨基酸残基上与它们不同。共有序列显示与其他微生物的漆酶有明显的同源性。
