Anh Dau Hung, Ullrich René, Benndorf Dirk, Svatos Ales, Muck Alexander, Hofrichter Martin
International Graduate School (IHI) Zittau, Unit of Environmental Biotechnology, Markt 23, 02763 Zittau, Germany.
Appl Environ Microbiol. 2007 Sep;73(17):5477-85. doi: 10.1128/AEM.00026-07. Epub 2007 Jun 29.
Coprophilous and litter-decomposing species (26 strains) of the genus Coprinus were screened for peroxidase activities by using selective agar plate tests and complex media based on soybean meal. Two species, Coprinus radians and C. verticillatus, were found to produce peroxidases, which oxidized aryl alcohols to the corresponding aldehydes at pH 7 (a reaction that is typical for heme-thiolate haloperoxidases). The peroxidase of Coprinus radians was purified to homogeneity and characterized. Three fractions of the enzyme, CrP I, CrP II, and CrP III, with molecular masses of 43 to 45 kDa as well as isoelectric points between 3.8 and 4.2, were identified after purification by anion-exchange and size exclusion chromatography. The optimum pH of the major fraction (CrP II) for the oxidation of aryl alcohols was around 7, and an H2O2 concentration of 0.7 mM was most suitable regarding enzyme activity and stability. The apparent Km values for ABTS [2,2'-azinobis(3-ethylbenzthiazolinesulfonic acid)], 2,6-dimethoxyphenol, benzyl alcohol, veratryl alcohol, and H2O2 were 49, 342, 635, 88, and 1,201 microM, respectively. The N terminus of CrP II showed 29% and 19% sequence identity to Agrocybe aegerita peroxidase (AaP) and chloroperoxidase, respectively. The UV-visible spectrum of CrP II was highly similar to that of resting-state cytochrome P450 enzymes, with the Soret band at 422 nm and additional maxima at 359, 542, and 571 nm. The reduced carbon monoxide complex showed an absorption maximum at 446 nm, which is characteristic of heme-thiolate proteins. CrP brominated phenol to 2- and 4-bromophenols and selectively hydroxylated naphthalene to 1-naphthol. Hence, after AaP, CrP is the second extracellular haloperoxidase-peroxygenase described so far. The ability to extracellularly hydroxylate aromatic compounds seems to be the key catalytic property of CrP and may be of general significance for the biotransformation of poorly available aromatic substances, such as lignin, humus, and organopollutants in soil litter and dung environments. Furthermore, aromatic peroxygenation is a promising target of biotechnological studies.
通过使用选择性琼脂平板试验和基于豆粕的复合培养基，对鬼伞属的嗜粪和分解落叶的物种（26株菌株）进行了过氧化物酶活性筛选。发现两个物种，辐射鬼伞和轮纹鬼伞，能够产生过氧化物酶，它们在pH 7时将芳醇氧化为相应的醛（这是血红素硫醇盐卤过氧化物酶的典型反应）。对辐射鬼伞的过氧化物酶进行了纯化并进行了表征。通过阴离子交换和尺寸排阻色谱纯化后，鉴定出该酶的三个组分，即CrP I、CrP II和CrP III，其分子量为43至45 kDa，等电点在3.8至4.2之间。主要组分（CrP II）氧化芳醇的最适pH约为7，就酶活性和稳定性而言，0.7 mM的H2O2浓度最为合适。ABTS [2,2'-偶氮二（3-乙基苯并噻唑啉磺酸）]、2,6-二甲氧基苯酚、苯甲醇、藜芦醇和H2O2的表观Km值分别为49、342、635、88和1201 microM。CrP II的N末端与盖囊侧耳过氧化物酶（AaP）和氯过氧化物酶的序列同一性分别为29%和19%。CrP II的紫外可见光谱与静止态细胞色素P450酶的光谱高度相似，Soret带在422 nm，在359、542和571 nm处还有其他最大值。还原态一氧化碳复合物在446 nm处有一个吸收最大值，这是血红素硫醇盐蛋白的特征。CrP将苯酚溴化为2-溴苯酚和4-溴苯酚，并将萘选择性羟基化为1-萘酚。因此，继AaP之后，CrP是迄今为止描述的第二种细胞外卤过氧化物酶-过氧合酶。细胞外羟基化芳香化合物的能力似乎是CrP的关键催化特性，对于土壤落叶和粪便环境中难利用的芳香物质，如木质素、腐殖质和有机污染物的生物转化可能具有普遍意义。此外，芳香过氧合是生物技术研究的一个有前景的目标。
