Purification and Characterization of Cellobiose Dehydrogenases from the White Rot Fungus Trametes versicolor
Purification and Characterization of Cellobiose Dehydrogenases from the White Rot Fungus Trametes versicolor
复制标题
白腐真菌栓菌中纤维二糖脱氢酶的纯化和表征
DOI：
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发表时间：
1996
影响因子：
4.4
通讯作者：
F. Archibald
中科院分区：
文献类型：
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作者：
B. Roy;T. Dumonceaux;A. Koukoulas;F. Archibald
白色腐烂真菌变色栓菌（Trametesversicolor）至少部分地通过经由许多分泌的氧化酶和过氧化酶氧化木质素来降解木质纤维素材料。一种细胞外还原酶，纤维二糖脱氢酶（CDH），氧化纤维二糖并还原不溶性Mn（IV）O（inf 2），通常在腐烂的木材中发现为深色沉积物，形成Mn（III），一种强大的木质素氧化剂。CDH还还原邻醌并产生糖酸，糖酸可促进锰过氧化物酶并因此促进木质素分解活性。为了更好地理解CDH在木质素降解中的作用，从T.变色。分离出两种不同的蛋白质;蛋白质的表观分子量分别为97，000和81，000，等电点分别为4.2和6.4。较大的CDH（CDH 4.2）含有黄素和血红素辅因子，而较小的CDH仅含有黄素（CDH 6.4）。这些CDH酶被纤维二糖和乳糖迅速还原，而被纤维素和某些纤维低聚糖稍微缓慢一些。这两种糖蛋白能够减少非常广泛的醌类和有机自由基物种，但不同的能力，以减少金属离子络合物。CDH 4.2的最适温度和pH受还原底物的影响。虽然CDH 4.2在邻醌类中表现出相当高的底物特异性，但它也可以快速还原结构上非常多样化的其他物种，从带负电荷的三碘离子到带正电荷的六水合铁离子。对于所有测试的底物，CDH 6.4显示出比CDH 4.2更高的K（infm）和更低的V（infmax）和周转数。此外，CDH 6.4不会在可能与生理相关的浓度下还原过渡金属Fe（III）、Cu（II）和Mn（III），而CDH 4.2能够快速还原甚至非常低浓度的这些离子。通过CDH 4.2还原Fe（III）和Cu（II）在维持芬顿型反应中可能是重要的，该反应产生可以裂解木质素和纤维素的羟基自由基。与来自黄孢原毛平革菌的CDH蛋白不同，CDH 4.2和CDH 6.4不能产生过氧化氢。
The white rot fungus Trametes versicolor degrades lignocellulosic material at least in part by oxidizing the lignin via a number of secreted oxidative and peroxidative enzymes. An extracellular reductive enzyme, cellobiose dehydrogenase (CDH), oxidizes cellobiose and reduces insoluble Mn(IV)O(inf2), commonly found as dark deposits in decaying wood, to form Mn(III), a powerful lignin-oxidizing agent. CDH also reduces ortho-quinones and produces sugar acids which can promote manganese peroxidase and therefore ligninolytic activity. To better understand the role of CDH in lignin degradation, proteins exhibiting cellobiose-dependent quinone-reducing activity were isolated and purified from cultures of T. versicolor. Two distinct proteins were isolated; the proteins had apparent molecular weights of 97,000 and 81,000 and isoelectric points of 4.2 and 6.4, respectively. The larger CDH (CDH 4.2) contained both flavin and heme cofactors, whereas the smaller contained only a flavin (CDH 6.4). These CDH enzymes were rapidly reduced by cellobiose and lactose and somewhat more slowly by cellulose and certain cello-oligosaccharides. Both glycoproteins were able to reduce a very wide range of quinones and organic radical species but differed in their ability to reduce metal ion complexes. Temperature and pH optima for CDH 4.2 were affected by the reduced substrate. Although CDH 4.2 showed rather high substrate specificity among the ortho-quinones, it could also rapidly reduce a structurally very diverse collection of other species, from negatively charged triiodide ions to positively charged hexaquo ferric ions. CDH 6.4 showed a higher K(infm) and a lower V(infmax) and turnover number than did CDH 4.2 for all substrates tested. Furthermore, CDH 6.4 did not reduce the transition metals Fe(III), Cu(II), and Mn(III) at concentrations likely to be physiologically relevant, while CDH 4.2 was able to rapidly reduce even very low concentrations of these ions. The reduction of Fe(III) and Cu(II) by CDH 4.2 may be important in sustaining a Fenton's-type reaction, which produces hydroxyl radicals that can cleave both lignin and cellulose. Unlike the CDH proteins from Phanerochaete chrysosporium, CDH 4.2 and CDH 6.4 are unable to produce hydrogen peroxide.