Unspecific peroxygenases (UPOs) constitute a new family of fungal heme-thiolate enzymes in which there is high biotechnological interest. Although several thousand genes encoding hypothetical UPO-type proteins have been identified in sequenced fungal genomes and other databases, only a few UPO enzymes have been experimentally characterized to date. Therefore, gene screening and heterologous expression from genetic databases are a priority in the search for ad hoc UPOs for oxyfunctionalization reactions of interest. Very recently, Escherichia coli production of a previously described basidiomycete UPO (as a soluble and active enzyme) has been reported. Here, we explored this convenient heterologous expression system to obtain the protein products from available putative UPO genes. In this way, two UPOs from the ascomycetes Collariella virescens (syn., Chaetomium virescens) and Daldinia caldariorum were successfully obtained, purified, and characterized. Comparison of their kinetic constants for oxidation of model substrates revealed 10- to 20-fold-higher catalytic efficiency of the latter enzyme in oxidizing simple aromatic compounds (such as veratryl alcohol, naphthalene, and benzyl alcohol). Homology molecular models of these enzymes showed three conserved and two differing residues in the distal side of the heme (the latter representing two different positions of a phenylalanine residue). Interestingly, replacement of the C. virescens UPO Phe88 by the homologous residue in the D. caldariorum UPO resulted in an F88L variant with 5- to 21-fold-higher efficiency in oxidizing these aromatic compounds.

IMPORTANCE UPOs catalyze regio- and stereoselective oxygenations of both aromatic and aliphatic compounds. Similar reactions were previously described for cytochrome P450 monooxygenases, but UPOs have the noteworthy biotechnological advantage of being stable enzymes requiring only H₂O₂ to be activated. Both characteristics are related to the extracellular nature of UPOs as secreted proteins. In the present study, the limited repertoire of UPO enzymes available for organic synthesis and other applications is expanded with the description of two new ascomycete UPOs obtained by Escherichia coli expression of the corresponding genes as soluble and active enzymes. Moreover, directed mutagenesis in E. coli, together with enzyme molecular modeling, provided relevant structure-function information on aromatic substrate oxidation by these two new biocatalysts.

非特异性过氧化酶（UPOs）构成了新的真菌血红素硫醇酯酶家族，在生物技术方面具有很高的兴趣。尽管已在测序的真菌基因组和其他数据库中鉴定了数千种假设的UPO型蛋白编码基因，但迄今为止，只有少数UPO酶在实验上得到了表征。因此，从遗传数据库进行基因筛选和异源表达是寻找感兴趣的氧官能化反应的特殊UPO的优先事项。最近，大肠杆菌已经报道了先前描述的担子菌UPO（作为可溶性和活性酶）的生产。在这里，我们探索了这种方便的异源表达系统，以从可用的假定的UPO基因获得蛋白质产物。通过这种方式，来自子囊菌（Collariella virescens）（合发，Chaetomium v​​irescens）和Daldinia caldariorum的两个UPO成功获得，纯化和表征。比较它们对模型底物的氧化动力学常数，发现后者在氧化简单的芳香族化合物（如藜芦醇，萘和苄醇）时的催化效率高10到20倍。这些酶的同源分子模型在血红素的远端显示了三个保守和两个不同的残基（后者代表苯丙氨酸残基的两个不同位置）。有趣的是，用D. caldariorum UPO中的同源残基替代了C. virescens UPO Phe88，导致F88L变体在氧化这些芳族化合物时的效率提高了5到21倍。

重要事项UPO催化芳族和脂族化合物的区域和立体选择性氧化。先前已经针对细胞色素P450单加氧酶描述了类似的反应，但是UPO具有值得注意的生物技术优势，因为它是仅需激活H ₂ O _2的稳定酶。这两个特征都与UPO作为分泌蛋白的细胞外特性有关。在本研究中，描述了通过大肠杆菌将相应基因表达为可溶性和活性酶而获得的两种新的子囊菌UPO，扩大了可用于有机合成和其他应用的UPO酶的种类。此外，在大肠杆菌中进行定向诱变与酶分子建模一起，提供了有关这两种新生物催化剂对芳香族底物氧化的相关结构功能信息。