Eggert C, Temp U, Eriksson K E
Department of Biochemistry and Molecular Biology, University of Georgia, Athens 30602-7229, USA.
Appl Environ Microbiol. 1996 Apr;62(4):1151-8. doi: 10.1128/aem.62.4.1151-1158.1996.
The white rot fungus Pycnoporus cinnabarinus was characterized with respect to its set of extracellular phenoloxidases. Laccase was produced as the predominant extracellular phenoloxidase in conjunction with low amounts of an unusual peroxidase. Neither lignin peroxidase nor manganese peroxidase was detected. Laccase was produced constitutively during primary metabolism. Addition of the most effective inducer, 2,5-xylidine, enhanced laccase production ninefold without altering the isoenzyme pattern of the enzyme. Laccase purified to apparent homogeneity was a single polypeptide having a molecular mass of approximately 81,000 Da, as determined by calibrated gel filtration chromatography, and a carbohydrate content of 9%. The enzyme displayed an unusual behavior on isoelectric focusing gels; the activity was split into one major band (pI, 3.7) and several minor bands of decreasing intensity which appeared at regular, closely spaced intervals toward the alkaline end of the gel. Repeated electrophoresis of the major band under identical conditions produced the same pattern, suggesting that the laccase was secreted as a single acidic isoform with a pI of about 3.7 and that the multiband pattern was an artifact produced by electrophoresis. This appeared to be confirmed by N-terminal amino acid sequencing of the purified enzyme, which yielded a single sequence for the first 21 residues. Spectroscopic analysis indicated a typical laccase active site in the P. cinnabarinus enzyme since all three typical Cu(II)-type centers were identified. Substrate specificity and inhibitor studies also indicated the enzyme to be a typical fungal laccase. The N-terminal amino acid sequence of the P. cinnabarinus laccase showed close homology to the N-terminal sequences determined for laccases from Trametes versicolor, Coriolus hirsutus, and an unidentified basidiomycete, PM1. The principal features of the P. cinnabarinus enzyme system, a single predominant laccase and a lack of lignin- or manganese-type peroxidase, make this organism an interesting model for further studies of possible alternative pathways of lignin degradation by white rot fungi.
对朱红密孔菌这种白腐真菌的胞外酚氧化酶系进行了表征。漆酶是主要产生的胞外酚氧化酶，同时还产生少量不同寻常的过氧化物酶。未检测到木质素过氧化物酶和锰过氧化物酶。漆酶在初级代谢过程中组成型产生。添加最有效的诱导剂2,5 - 二甲基苯胺可使漆酶产量提高9倍，且不改变该酶的同工酶模式。经校准凝胶过滤色谱法测定，纯化至表观均一的漆酶是一条单一多肽链，分子量约为81,000 Da，碳水化合物含量为9%。该酶在等电聚焦凝胶上表现出不同寻常的行为；其活性被分离成一条主要条带（pI为3.7）和几条强度逐渐降低的次要条带，这些次要条带出现在凝胶碱性端方向规则且间隔紧密的位置。在相同条件下对主要条带进行重复电泳产生相同的模式，这表明漆酶是以单一酸性同工型分泌的，pI约为3.7，多带模式是电泳产生的假象。纯化酶的N端氨基酸测序似乎证实了这一点，其前21个残基产生了单一序列。光谱分析表明朱红密孔菌的漆酶中存在典型的漆酶活性位点，因为鉴定出了所有三个典型的Cu(II)型中心。底物特异性和抑制剂研究也表明该酶是典型的真菌漆酶。朱红密孔菌漆酶的N端氨基酸序列与云芝、毛云芝和一种未鉴定的担子菌PM1的漆酶N端序列显示出密切的同源性。朱红密孔菌酶系统的主要特征——单一主要漆酶以及缺乏木质素或锰型过氧化物酶，使得该生物体成为进一步研究白腐真菌可能的木质素降解替代途径的有趣模型。
