The ligninolytic system of the white rot fungus Pycnoporus cinnabarinus: Purification and characterization of the laccase
The ligninolytic system of the white rot fungus Pycnoporus cinnabarinus: Purification and characterization of the laccase
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DOI：
10.1128/aem.62.4.1151-1158.1996
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发表时间：
1996-04-01
影响因子：
4.4
通讯作者：
Eriksson, KEL
中科院分区：
文献类型：
--
作者：
Eggert, C;Temp, U;Eriksson, KEL
研究了白色腐真菌朱砂密孔菌（Pycnoporus cinnabarinus）胞外酚氧化酶的特性。漆酶作为主要的细胞外酚氧化酶与少量的不寻常的过氧化物酶一起产生。漆酶在初级代谢过程中组成型产生，加入最有效的诱导剂2，5-二甲基苯胺，使漆酶产量提高9倍，而不改变酶的同工酶谱，纯化至表观均一性的漆酶是一种分子量约为81，000 Da的单一多肽，碳水化合物含量为9%。该酶在等电聚焦凝胶上表现出不寻常的行为;活动分为一个主要波段（pI，3.7）和几条强度递减的次带，它们以规则的、紧密间隔出现在凝胶的碱性端。在相同条件下重复电泳主带产生相同的图谱，这表明漆酶是作为单一酸性同工型分泌的，pi约为3.7，并且多带模式是由电泳产生的假象。这似乎被纯化的酶的N-末端氨基酸测序所证实，其产生了前21个残基的单一序列。光谱分析表明，由于所有三个典型的Cu（II）型中心被鉴定，所以在朱砂叶甲酶中存在典型的漆酶活性位点。底物特异性和抑制剂研究也表明该酶是一种典型的真菌漆酶。该酶的N-末端氨基酸序列与Trametes versicolor、Coriolus hirsutus和一种未鉴定的担子菌PM 1的漆酶的N-末端序列具有很高的同源性。P. cinnabarinus酶系统的主要特征是单一的优势漆酶和缺乏木质素或锰型过氧化物酶，这使得该生物体成为进一步研究白色腐真菌降解木质素的可能替代途径的有趣模型。
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.