Purification and characterization of cellobiose dehydrogenase from the plant pathogen Sclerotium (Athelia) rolfsii
Purification and characterization of cellobiose dehydrogenase from the plant pathogen Sclerotium (Athelia) rolfsii
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DOI：
10.1128/aem.67.4.1766-1774.2001
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发表时间：
2001-04-01
影响因子：
4.4
通讯作者：
Haltrich, D
中科院分区：
文献类型：
--
作者：
Baminger, U;Subramaniam, SS;Haltrich, D
纤维素二糖脱氢酶（CDH）是由几种木材降解真菌产生的细胞外血黄酶。在合适的电子受体存在下，例如2,6-二氯吲哚酚（DCIP）、细胞色素c或金属离子，CDH将纤维二糖氧化为纤维生物内酯。植物病原真菌罗尔夫菌核菌（远形菌：Athelia rolfsii）菌株CBS 191,62在含纤维素的培养基上生长时产生显著高水平的CDH活性。在每升形成的7,500 U胞外酶活性中，不到10%可归因于蛋白水解产物纤维素二糖：醌氧化还原酶。与木材腐烂真菌CDH一样，来自S. rolfsii的完整单体酶每个分子含有一个血红素b和一个黄素腺嘌呤二核苷酸辅因子。其分子大小为101 kDa，其中15%为糖基化，pi值为4.2。优选底物为纤维素二糖和纤维素低聚糖；此外，对乳糖、硫纤维素二糖和木糖二糖被有效氧化。细胞色素c（马）和氮基二-（3-乙基-苯并噻唑-6-磺酸）阳离子自由基是最佳的电子受体，DCIP、1,4-苯并醌、亚甲基蓝等吩噻嗪类染料、梅多拉蓝等吩恶嗪类染料和铁氰化物也是最佳的电子受体。另外，电子可以转移到氧上。用木瓜蛋白酶进行有限的体外蛋白水解，形成了几个对DCIP有活性但对细胞色素c没有活性的蛋白片段。这样一个含有黄素的片段，质量为75 kDa， pI为5.1，缺乏血红素结构域，被分离出来并部分表征。
Cellobiose dehydrogenase (CDH) is an extracellular hemoflavoenzyme produced by several wood-degrading fungi. In the presence of a suitable electron acceptor, e.g., 2,6-dichloro-indophenol (DCIP), cytochrome c, or metal ions, CDH oxidizes cellobiose to cellobionolactone. The phytopathogenic fungus Sclerotium rolfsii (teleomorph: Athelia rolfsii) strain CBS 191,62 produces remarkably high levels of CDH activity when grown on a cellulose-containing medium. Of the 7,500 U of extracellular enzyme activity formed per liter, less than 10% can be attributed to the proteolytic product cellobiose:quinone oxidoreductase. As with CDH hom wood-rotting fungi, the intact, monomeric enzyme from S. rolfsii contains one heme b and one flavin adenine dinucleotide cofactor per molecule. It has a molecular size of 101 kDa, of which 15% is glycosylation, and a pi value of 4.2. The preferred substrates are cellobiose and cellooligosaccharides; additionally, p-lactose, thiocellobiose, and xylobiose are efficiently oxidized. Cytochrome c (equine) and the azino-di-(3-ethyl-benzthiazolin-6-sulfoic acid) cation radical were the best electron accepters, while DCIP, 1,4-benzoquinone, phenothiazine dyes such as methylene blue, phenoxazine dyes such as Metdola's blue, and ferricyanide were also excellent accepters. In addition, electrons can be transferred to oxygen. Limited in vitro proteolysis with papain resulted in the formation of several protein fragments that are active with DCIP but not with cytochrome c. Such a flavin-containing fragment, with a mass of 75 kDa and a pI of 5.1 and lacking the heme domain, was isolated and partially characterized.