TY - GEN A1 - Ullrich, René A1 - Nüske, Jörg A1 - Scheibner, Katrin A1 - Spantzel, Jörg A1 - Hofrichter, Martin T1 - Novel Haloperoxidase from the Agaric Basidiomycete Agrocybe aegerita Oxidizes Aryl Alcohols and Aldehydes T2 - Applied and Environmental Microbiology N2 - Agrocybe aegerita, a bark mulch- and wood-colonizing basidiomycete, was found to produce a peroxidase (AaP) that oxidizes aryl alcohols, such as veratryl and benzyl alcohols, into the corresponding aldehydes and then into benzoic acids. The enzyme also catalyzed the oxidation of typical peroxidase substrates, such as 2,6-dimethoxyphenol (DMP) or 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonate) (ABTS). A. aegerita peroxidase production depended on the concentration of organic nitrogen in the medium, and highest enzyme levels were detected in the presence of soybean meal. Two fractions of the enzyme, AaP I and AaP II, which had identical molecular masses (46 kDa) and isoelectric points of 4.6 to 5.4 and 4.9 to 5.6, respectively (corresponding to six different isoforms), were identified after several steps of purification, including anion- and cation-exchange chromatography. The optimum pH for the oxidation of aryl alcohols was found to be around 7, and the enzyme required relatively high concentrations of H2O2 (2 mM) for optimum activity. The apparent Km values for ABTS, DMP, benzyl alcohol, veratryl alcohol, and H2O2 were 37, 298, 1,001, 2,367 and 1,313 μM, respectively. The N-terminal amino acid sequences of the main AaP II spots blotted after two-dimensional gel electrophoresis were almost identical and exhibited almost no homology to the sequences of other peroxidases from basidiomycetes, but they shared the first three amino acids, as well as two additional amino acids, with the heme chloroperoxidase (CPO) from the ascomycete Caldariomyces fumago. This finding is consistent with the fact that AaP halogenates monochlorodimedone, the specific substrate of CPO. The existence of haloperoxidases in basidiomycetous fungi may be of general significance for the natural formation of chlorinated organic compounds in forest soils. KW - Agrocybe aegerita KW - Peroxygenase KW - Peroxidase KW - Basidiomycete Y1 - 2004 UR - http://aem.asm.org/content/70/8/4575.long U6 - https://doi.org/10.1128/AEM.70.8.4575-4581.2004 SN - 1098-5336 VL - 70 IS - 8 SP - 4575 EP - 4581 ER - TY - GEN A1 - Stritter, Benjamin A1 - Freiling, Felix A1 - König, Hartmut A1 - Rietz, René A1 - Ullrich, Steffen A1 - Gernler, Alexander von A1 - Erlacher, Felix T1 - Cleaning up Web 2.0's Security Mess - at Least Partly T2 - IEEE Security & Privacy Y1 - 2016 UR - http://online.qmags.com/SNP0316?pg=92&mode=2#pg92&mode2 SN - 1540-7993 VL - 14 IS - 2 SP - 48 EP - 71 ER - TY - GEN A1 - Babot, Esteban D. A1 - Río, José C. del A1 - Cañellas, Marina A1 - Sancho, Ferran A1 - Lucas, Fátima A1 - Guallar, Víctor A1 - Kalum, Lisbeth A1 - Lund, Henrik A1 - Gröbe, Glenn A1 - Scheibner, Katrin A1 - Ullrich, René A1 - Hofrichter, Martin A1 - Martínez, Angel T. A1 - Gutiérrez, Ana T1 - Steroid hydroxylation by basidiomycete peroxygenases: A combined experimental and computational study T2 - Applied and Environmental Microbiology N2 - The goal of this study is the selective oxyfunctionalization of steroids under mild and environmentally-friendly conditions using fungal enzymes. With this purpose, peroxygenases from three basidiomycete species were tested for hydroxylation of a variety of steroidal compounds, using H2O2 as the only cosubstrate. Two of them are wild-type enzymes from Agrocybe aegerita and Marasmius rotula, and the third one is a recombinant enzyme from Coprinopsis cinerea. The enzymatic reactions on free and esterified sterols, and steroid hydrocarbons and ketones were followed by gas chromatography, and the products were identified by mass spectrometry. Hydroxylation at the side chain over the steroidal rings was preferred, with the 25-hydroxyderivatives predominating (interestingly antiviral and other biological activities of 25-hydroxycholesterol have been recently reported). However, hydroxylation in the ring moiety and terminal hydroxylation at the side-chain was also observed in some steroids, the former favored by the absence of oxygenated groups at C3 and by the presence of conjugated double bonds in the rings. To understand the yield and selectivity differences between the different steroids, a computational study was performed using Protein Energy Landscape Exploration (PELE) software for dynamic ligand diffusion. These simulations showed that the active site geometry and hydrophobicity favors the entrance of the steroid side-chain, while the entrance of the ring is energetically penalized. Also, a direct correlation between the conversion rate and the side-chain entrance ratio could be established, that explains the varying reaction yields observed. KW - Peroxyenase Y1 - 2015 UR - http://aem.asm.org/content/early/2015/04/08/AEM.00660-15 U6 - https://doi.org/10.1128/AEM.00660-15 SN - 0099-2240 SN - 1098-5336 VL - 81 IS - 12 SP - 4130 EP - 4142 ER - TY - GEN A1 - Poraj-Kobielska, Marzena A1 - Peter, Sebastian A1 - Leonhardt, Sabrina A1 - Ullrich, René A1 - Scheibner, Katrin A1 - Hofrichter, Martin T1 - Immobilization of unspecific peroxygenases (EC 1.11.2.1) in PVA/PEG gel and hollow fiber modules T2 - Biochemical Engineering Journal N2 - The immobilization of enzymes has many advantages, such as higher stability, easier handling, and reuse of the catalyst. Here we report, for the first time, two effective methods for the immobilization of unspecific peroxygenase (UPO; EC 1.11.2.1). This biocatalyst type comprises heavily glycosylated heme-thiolate proteins that catalyze various biotechnologically relevant oxyfunctionalizations. Both the encapsulation in cryogel and the retention of the enzyme in hollow fiber modules were found to be efficient methods for their immobilization. After encapsulation, the enzyme still exhibited 60% of its initial activity. Interestingly, we did not find differences in the kinetic parameters of free and immobilized UPOs. In long-term experiments, the conversion of the pharmaceutical diclofenac with immobilized UPOs in different reactor types yielded between 62 mg and 154 mg of the major human drug metabolite 4′-hydroxydiclofenac. The maximal total turnover number was about 60-fold higher compared to the free enzyme. A test over 5 months showed that storage of encapsulated UPOs in non-polar solvents (e.g., cyclohexane) helps to preserve the enzyme stability and increases their relative activity (by about ∼150%, in the case of diclofenac hydroxylation). In addition to the hydrophilic substrate diclofenac, encapsulated UPOs also oxidized the hydrophobic model compound cyclohexane. KW - Peroxygenase Y1 - 2015 UR - http://www.sciencedirect.com/science/article/pii/S1369703X15000820 U6 - https://doi.org/10.1016/j.bej.2015.02.037 SN - 1369-703X VL - 98 SP - 144 EP - 150 ER - TY - GEN A1 - Kiebist, Jan A1 - Schmidtke, Kai-Uwe A1 - Zimmermann, Jörg A1 - Kellner, Harald A1 - Jehmlich, Nico A1 - Ullrich, René A1 - Zänder, Daniel A1 - Hofrichter, Martin A1 - Scheibner, Katrin T1 - A peroxygenase from Chaetomium globosum catalyzes the selective oxygenation of testosterone T2 - ChemBioChem N2 - Unspecific peroxygenases (UPO, EC 1.11.2.1) secreted by fungi open an efficient way to selectively oxyfunctionalize diverse organic substrates including less activated hydrocarbons by transferring peroxide-borne oxygen. Herein, we investigated a cell-free approach to incorporate epoxy and hydroxyl functionalities directly into the bulky molecule of testosterone by a novel unspecific peroxygenase that was produced by the ascomycetous fungus Chaetomium globosum in a complex medium rich in carbon and nitrogen. Purification by fast protein liquid chromatography revealed two enzyme fractions with the same molecular mass of 36 kDa and specific activities of 4.4 to 12 U mg-1. Whereas well-known UPOs of Agrocybe aegerita (AaeUPO) and Marasmius rotula (MroUPO) failed to convert testosterone in a comparative study, the UPO of C. globosum (CglUPO) accepted testosterone as substrate and converted it with up to 7,000 total turnovers (TTN) into two oxygenated products: the 4,5-epoxide of testosterone in β-configuration and 16α-hydroxytestosterone. The reaction was performed at 100-mg scale resulting in the formation of about 90 % of the epoxide and 10 % of the hydroxylation product, which both could be isolated with purities above 96 %. Thus, CglUPO may be a promising biocatalyst for the oxyfunctionalization of bulky steroids and provide a useful tool for the synthesis of pharmaceutically relevant steroidal molecules. KW - peroxidase KW - hydroxylation Y1 - 2017 UR - http://onlinelibrary.wiley.com/doi/10.1002/cbic.201600677/abstract U6 - https://doi.org/10.1002/cbic.201600677 SN - 1439-7633 VL - 18 IS - 6 SP - 563 EP - 569 ER - TY - GEN A1 - Karich, Alexander A1 - Scheibner, Katrin A1 - Ullrich, René A1 - Hofrichter, Martin T1 - Exploring the catalase activity of unspecific peroxygenases and the mechanism of peroxide-dependent heme destruction T2 - Journal of Molecular Catalysis B: Enzymatic N2 - The catalase activity of three unspecific peroxygenases (UPOs) from the agaric basidiomycetes Agrocybe aegerita, Coprinopsis cinerea and Marasmius rotula was investigated. The study included analysis of pH dependency of the catalase reaction and H₂O₂ mediated enzyme inactivation as well as experiments on the influence of a second substrate on the course of catalase reaction. Apparent kinetic parameters (Km, kcat) for the catalase activity of UPOs were determined. Inactivation of UPOs by H₂O₂ is discussed with regard to O₂ production and remaining UPO activity. Furthermore formation of biliverdin as heme destruction product was demonstrated along with the formation of UPO compound III as a possible intermediate that forces the destruction process. Radical trapping experiments with methyl benzoate gave indication for the formation of hydroxyl radicals in the presence of excess H₂O₂. Eventually, a plausible pathway of heme destruction has been proposed, proceeding via UPO compound III and subsequent hydroxyl radical formation, which in turn may cause heme bleaching and verdoheme and biliverdin formation. KW - Peroxygenase Y1 - 2016 UR - http://www.sciencedirect.com/science/article/pii/S1381117716302090 U6 - https://doi.org/10.1016/j.molcatb.2016.10.014 SN - 1381-1177 VL - 134 IS - A SP - 238 EP - 246 ER - TY - CHAP A1 - Rietz, René A1 - König, Hartmut A1 - Ullrich, Steffen A1 - Stritter, Benjamin T1 - Firewalls for the Web 2.0 T2 - Proceedings of the 2016 IEEE International Conference on Software Quality, Reliability and Security (QRS), 1-3 Aug. 2016 N2 - The widespread use of Web 2.0 technologies yields an increasing threat potential for users and related systems. Modern web applications and online services are nowadays based on Web 2.0 technologies, such as JavaScript and AJAX, and thus on the execution of active content in the browsers of the users. Firewalls are a common practice to securely connecting to the internet. In this paper, we propose a novel perimeter firewall architecture for web applications that addresses the entire process chain starting from the data transfer with HTTP via the analysis of manipulated web documents to the extraction and analysis of active contents. The basic idea is to allow only a restricted set of web applications to pass the firewall based on a model of their HTML and JavaScript structure. We evaluate the capability of the resulting models for identifying the underlying web applications and their ability to ward off additional malicious inputs. Y1 - 2016 UR - http://ieeexplore.ieee.org/document/7589804/ SN - 978-1-5090-4127-5 SN - 978-1-5090-4128-2 U6 - https://doi.org/10.1109/QRS.2016.36 SP - 242 EP - 253 PB - IEEE CY - Piscataway, NJ ER - TY - GEN A1 - Karich, Alexander A1 - Ullrich, René A1 - Scheibner, Katrin A1 - Hofrichter, Martin T1 - Fungal unspecific peroxygenases oxidize the majority of organic EPA priority pollutants T2 - Frontiers in Microbiology N2 - Unspecific peroxygenases (UPOs) are secreted fungal enzymes with promiscuity for oxygen transfer and oxidation reactions. Functionally, they represent hybrids of P450 monooxygenases and heme peroxidases; phylogenetically they belong to the family of heme-thiolate peroxidases. Two UPOs from the basidiomycetous fungi Agrocybe aegerita (AaeUPO) and Marasmius rotula (MroUPO) converted 35 out of 40 compounds listed as EPA priority pollutants, including chlorinated benzenes and their derivatives, halogenated biphenyl ethers, nitroaromatic compounds, polycyclic aromatic hydrocarbons (PAHs) and phthalic acid derivatives. These oxygenations and oxidations resulted in diverse products and-if at all-were limited for three reasons: (i) steric hindrance caused by multiple substitutions or bulkiness of the compound as such (e.g., hexachlorobenzene or large PAHs), (ii) strong inactivation of aromatic rings (e.g., nitrobenzene), and (iii) low water solubility (e.g., complex arenes). The general outcome of our study is that UPOs can be considered as extracellular counterparts of intracellular monooxygenases, both with respect to catalyzed reactions and catalytic versatility. Therefore, they should be taken into consideration as a relevant biocatalytic detoxification and biodegradation tool used by fungi when confronted with toxins, xenobiotics and pollutants in their natural environments. KW - Peroxygenase Y1 - 2017 UR - https://www.frontiersin.org/articles/10.3389/fmicb.2017.01463/full U6 - https://doi.org/10.3389/fmicb.2017.01463 SN - 1664-302X VL - 8 ER - TY - GEN A1 - Olmedo, Andrés A1 - Río, José C. del A1 - Kiebist, Jan A1 - Ullrich, René A1 - Hofrichter, Martin A1 - Scheibner, Katrin A1 - Martínez, Angel T. A1 - Gutiérrez, Ana T1 - Fatty Acid Chain Shortening by a Fungal Peroxygenase T2 - Chemistry A European Journal N2 - A recently discovered peroxygenase from the fungus Marasmius rotula (MroUPO) is able to catalyze the progressive one-carbon shortening of medium and longchain mono- and dicarboxylic acids by itself alone, in the presence of H₂O₂. The mechanism, analyzed using H₂O, starts with an a-oxidation catalyzed by MroUPO generat- ing an α-hydroxy acid, which is further oxidized by the enzyme to a reactive α-keto intermediate whose decarboxylation yields the one-carbon shorter fatty acid. Compared with the previously characterized peroxygenase of Agrocybe aegerita, a wider heme access channel, enabling fatty acid positioning with the carboxylic end near the heme cofactor (as seen in one of the crystal structures available) could be at the origin of the unique ability of MroUPO shortening carboxylic acid chains. KW - Peroxygenase Y1 - 2017 U6 - https://doi.org/10.1002/chem.201704773 SN - 1521-3765 SN - 0947-6539 VL - 23 SP - 16989 EP - 67 ER - TY - GEN A1 - Martinez, Angel T. A1 - Ruiz-Duenas, Francisco J. A1 - Camarero, Susana A1 - Serrano, Ana A1 - Linde, Dolores A1 - Lund, Henrik A1 - Vind, Jesper A1 - Tovborg, Morton A1 - Herold-Majumdar, Owik M. A1 - Hofrichter, Martin A1 - Liers, Christiane A1 - Ullrich, René A1 - Scheibner, Katrin A1 - Sannia, Giovanni A1 - Piscitelli, Alessandra A1 - Sener, Mehmet E. A1 - Kilic, Sibel A1 - Berkel, Willem J. H. van A1 - Guallar, Víctor A1 - Lucas, Maria Fátima A1 - Zuhse, Ralf A1 - Ludwig, Roland A1 - Hollmann, Frank A1 - Fernández-Fueyo, Elena A1 - Record, Eric A1 - Faulds, Craig B. A1 - Tortajada, Marta A1 - Winckelmann, Ib A1 - Rasmussen, Jo-Anne A1 - Gelo-Pujic, Mirjana A1 - Gutiérrez, Ana A1 - Rio, José C. del A1 - Rencoret, Jorge A1 - Alcalde, Miguel T1 - Oxidoreductases on their way to industrial biotransformations T2 - Biotechnology Advances N2 - Fungi produce heme-containing peroxidases and peroxygenases, flavin-containing oxidases and dehydrogenases, and different copper-containing oxidoreductases involved in the biodegradation of lignin and other recalcitrant compounds. Heme peroxidases comprise the classical ligninolytic peroxidases and the new dye-decolorizing peroxidases, while heme peroxygenases belong to a still largely unexplored superfamily of heme-thiolate proteins. Nevertheless, basidiomycete unspecific peroxygenases have the highest biotechnological interest due to their ability to catalyze a variety of regio- and stereo-selective monooxygenation reactions with H2O2 as the source of oxygen and final electron acceptor. Flavo-oxidases are involved in both lignin and cellulose decay generating H2O2 that activates peroxidases and generates hydroxyl radical. The group of copper oxidoreductases also includes other H2O2 generating enzymes - copper-radical oxidases - together with classical laccases that are the oxidoreductases with the largest number of reported applications to date. However, the recently described lytic polysaccharide monooxygenases have attracted the highest attention among copper oxidoreductases, since they are capable of oxidatively breaking down crystalline cellulose, the disintegration of which is still a major bottleneck in lignocellulose biorefineries, along with lignin degradation. Interestingly, some flavin-containing dehydrogenases also play a key role in cellulose breakdown by directly/indirectly “fueling” electrons for polysaccharide monooxygenase activation. Many of the above oxidoreductases have been engineered, combining rational and computational design with directed evolution, to attain the selectivity, catalytic efficiency and stability properties required for their industrial utilization. Indeed, using ad hoc software and current computational capabilities, it is now possible to predict substrate access to the active site in biophysical simulations, and electron transfer efficiency in biochemical simulations, reducing in orders of magnitude the time of experimental work in oxidoreductase screening and engineering. What has been set out above is illustrated by a series of remarkable oxyfunctionalization and oxidation reactions developed in the frame of an intersectorial and multidisciplinary European RTD project. The optimized reactions include enzymatic synthesis of 1-naphthol, 25-hydroxyvitamin D3, drug metabolites, furandicarboxylic acid, indigo and other dyes, and conductive polyaniline, terminal oxygenation of alkanes, biomass delignification and lignin oxidation, among others. These successful case stories demonstrate the unexploited potential of oxidoreductases in medium and large-scale biotransformations. KW - Peroxygenase KW - Biotechnology Y1 - 2017 U6 - https://doi.org/10.1016/j.biotechadv.2017.06.003 SN - 1873-1899 SN - 0734-9750 VL - 35 IS - 6 SP - 815 EP - 831 ER - TY - GEN A1 - Ullrich, René A1 - Poraj-Kobielska, Marzena A1 - Scholze, Steffi A1 - Halbout, Claire A1 - Sandvoss, Martin A1 - Pecyna, Marek J. A1 - Scheibner, Katrin A1 - Hofrichter, Martin T1 - Side chain removal from corticosteroids by unspecific peroxygenase T2 - Journal of Inorganic Biochemistry N2 - Two unspecific peroxygenases (UPO, EC 1.11.2.1) from the basidiomycetous fungi Marasmius rotula and Marasmius wettsteinii oxidized steroids with hydroxyacetyl and hydroxyl functionalities at C17 - such as cortisone, Reichstein's substance S and prednisone - via stepwise oxygenation and final fission of the side chain. The sequential oxidation started with the hydroxylation of the terminal carbon (C21) leading to a stable geminal alcohol (e.g. cortisone 21-gem-diol) and proceeded via a second oxygenation resulting in the corresponding α-ketocarboxylic acid (e.g. cortisone 21-oic acid). The latter decomposed under formation of adrenosterone (4-androstene-3,11,17-trione) as well as formic acid and carbonic acid (that is in equilibrium with carbon dioxide); fission products comprising two carbon atoms such as glycolic acid or glyoxylic acid were not detected. Protein models based on the crystal structure data of MroUPO (Marasmius rotula unspecific peroxygenase) revealed that the bulky cortisone molecule suitably fits into the enzyme's access channel, which enables the heme iron to come in close contact to the carbons (C21, C20) of the steroidal side chain. ICP-MS analysis of purified MroUPO confirmed the presence of magnesium supposedly stabilizing the porphyrin ring system. KW - Peroxygenation KW - Peroxygenase KW - P450 KW - Deacylation Y1 - 2018 U6 - https://doi.org/10.1016/j.jinorgbio.2018.03.011 SN - 1873-3344 SN - 0162-0134 VL - 183 SP - 84 EP - 93 ER - TY - GEN A1 - Aranda, Carmen A1 - Ullrich, René A1 - Kiebist, Jan A1 - Scheibner, Katrin A1 - Río, José C. del A1 - Hofrichter, Martin A1 - Martínez, Angel T. A1 - Gutiérrez, Ana T1 - Selective synthesis of the resveratrol analogue 4,4′-dihydroxy-trans-stilbene and stilbenoids modification by fungal peroxygenases T2 - Catalysis Science & Technology N2 - This work gives first evidence that the unspecific peroxygenases (UPOs) from the basidiomycetes Agrocybe aegerita (AaeUPO), Coprinopsis cinerea (rCciUPO) and Marasmius rotula (MroUPO) are able to catalyze the regioselective hydroxylation of trans-stilbene to 4,4′-dihydroxy-trans-stilbene (DHS), a resveratrol (RSV) analogue whose preventive effects on cancer invasion and metastasis have very recently been shown. Nearly complete transformation of substrate (yielding DHS) was achieved with the three enzymes tested, using H2O2 as the only co-substrate, with AaeUPO showing exceptionally higher total turnover number (200 000) than MroUPO (26 000) and rCciUPO (1400). Kinetic studies demonstrated that AaeUPO was the most efficient enzyme catalyzing stilbene dihydroxylation with catalytic efficiencies (kcat/Km) one and two orders of magnitude higher than those of MroUPO and rCciUPO, so that 4-hydroxystilbene appears to be the best UPO substrate reported to date. In contrast, the peroxygenase from the ascomycete Chaetomium globosum (CglUPO) failed to hydroxylate trans-stilbene at the aromatic ring and instead produced the trans-epoxide in the alkenyl moiety. In addition, stilbenoids such as pinosylvin (Pin) and RSV were tested as substrates for the enzymatic synthesis of RSV from Pin and oxyresveratrol (oxyRSV) from both RSV and Pin. Overall, lower conversion rates and regioselectivities compared with trans-stilbene were accomplished by three of the UPOs, and no conversion was observed with CglUPO. The highest amount of RSV (63% of products) and oxyRSV (78%) were again attained with AaeUPO. True peroxygenase activity was demonstrated by incorporation of 18O from H218O2 into the stilbene hydroxylation products. Differences in the number of phenylalanine residues at the heme access channels seems related to differences in aromatic hydroxylation activity, since they would facilitate substrate positioning by aromatic-aromatic interactions. The only ascomycete UPO tested (that of C. globosum) turned out to have the most differing active site (distal side of heme cavity) and reactivity with stilbenes resulting in ethenyl epoxidation instead of aromatic hydroxylation. The above oxyfunctionalizations by fungal UPOs represent a novel and simple alternative to chemical synthesis for the production of DHS, RSV and oxyRSV. KW - Peroxygenase Y1 - 2018 U6 - https://doi.org/10.1039/C8CY00272J SN - 2044-4761 SN - 2044-4753 VL - 9 IS - 8 SP - 2394 EP - 2401 ER - TY - CHAP A1 - Hofrichter, Martin A1 - Kellner, Harald A1 - Herzog, Robert A1 - Karich, Alexander A1 - Liers, Christiane A1 - Scheibner, Katrin A1 - Kimani, Virginia Wambui A1 - Ullrich, René ED - Nevalainen, Helena T1 - Fungal Peroxygenases: A Phylogenetically Old Superfamily of Heme Enzymes with Promiscuity for Oxygen Transfer Reactions. T2 - Grand Challenges in Fungal Biotechnology KW - Peroxygenase Y1 - 2020 SN - 978-3-030-29540-0 SN - 978-3-030-29541-7 U6 - https://doi.org/10.1007/978-3-030-29541-7 SN - 2367-1017 SN - 2367-1025 SP - 369 EP - 403 PB - Springer Nature CY - Cham ET - 1. Auflage ER - TY - GEN A1 - Scheibner, Katrin A1 - Ullrich, René A1 - Kiebist, Jan A1 - Kellner, Harald A1 - Hofrichter, Martin T1 - Unspezifische Peroxygenasen - Oxyfunktionalisierung außerhalb der Pilzhyphe T2 - Biospektrum KW - Peroxygenase Y1 - 2020 U6 - https://doi.org/10.1007/s12268-020-1338-x SN - 1868-6249 SN - 0947-0867 VL - 26 IS - 1 SP - 103 EP - 106 ER - TY - GEN A1 - Kiebist, Jan A1 - Schmidtke, Kai-Uwe A1 - Schramm, Marina A1 - Hofrichter, Martin A1 - König, Rosalie A1 - Quint, Stephan A1 - Kohlmann, Johannes A1 - Zuhse, Ralf A1 - Ullrich, René A1 - Hofrichter, Martin A1 - Scheibner, Katrin T1 - Biocatalytic syntheses of antiplatelet metabolites of the thienopyridines clopidogrel and prasugrel using fungal peroxygenases T2 - Journal of Fungi N2 - Antithrombotic thienopyridines, such as clopidogrel and prasugrel, are prodrugs that undergo a metabolic two-step bioactivation for their pharmacological efficacy. In the first step, a thiolactone is formed, which is then converted by cytochrome P450-dependent oxidation via sulfenic acids to the active thiol metabolites. These metabolites are the active compounds that inhibit the platelet P2Y12 receptor and thereby prevent atherothrombotic events. Thus far, described biocatalytic and chemical synthesis approaches to obtain active thienopyridine metabolites are rather complex and suffer from low yields. In the present study, several unspecific peroxygenases (UPOs, EC 1.11.2.1) known to efficiently mimic P450 reactions in vitro—but requiring only hydroperoxide as oxidant—were tested for biocatalytic one-pot syntheses. In the course of the reaction optimization, various parameters such as pH and reductant, as well as organic solvent and amount were varied. The best results for the conversion of 1 mM thienopyridine were achieved using 2 U mL−1 of a UPO from agaric fungus Marasmius rotula (MroUPO) in a phosphate-buffered system (pH 7) containing 5 mM ascorbate, 2 mM h−1 H2O2 and 20% acetone. The preparation of the active metabolite of clopidogrel was successful via a two-step oxidation with an overall yield of 25%. In the case of prasugrel, a cascade of porcine liver esterase (PLE) and MroUPO was applied, resulting in a yield of 44%. The two metabolites were isolated with high purity, and their structures were confirmed by MS and MS2 spectrometry as well as NMR spectroscopy. The findings broaden the scope of UPO applications again and demonstrate that they can be effectively used for the selective synthesis of metabolites and late-state diversification of organic molecules, circumventing complex multistage chemical syntheses and providing sufficient material for structural elucidation, reference material, or cellular assays. KW - Peroxygenase KW - clopidogrel Y1 - 2021 UR - https://www.mdpi.com/2309-608X/7/9/752 U6 - https://doi.org/10.3390/jof7090752 SN - 2309-608X VL - 7 IS - 9 SP - 1 EP - 17 ER - TY - GEN A1 - Gomes de Santos, Patricia A1 - Hoang, Manh Dat A1 - Kiebist, Jan A1 - Kellner, Harald A1 - Ullrich, René A1 - Scheibner, Katrin A1 - Hofrichter, Martin A1 - Liers, Christiane A1 - Alcalde, Miguel T1 - Functional Expression of Two Unusual Acidic Peroxygenases from Candolleomyces aberdarensis in Yeasts by Adopting Evolved Secretion Mutations T2 - Applied and environmental microbiology N2 - Fungal unspecific peroxygenases (UPOs) are emergent biocatalysts that perform highly selective C-H oxyfunctionalizations of organic compounds, yet their heterologous production at high levels is required for their practical use in synthetic chemistry. Here, we achieved functional expression of two new unusual acidic peroxygenases from Candolleomyces (Psathyrella) aberdarensis (PabUPO) in yeasts and their production at a large scale in a bioreactor. Our strategy was based on adopting secretion mutations from an Agrocybe aegerita UPO mutant, the PaDa-I variant, designed by directed evolution for functional expression in yeast, which belongs to the same phylogenetic family as PabUPOs, long-type UPOs, and shares 65% sequence identity. After replacing the native signal peptides with the evolved leader sequence from PaDa-I, we constructed and screened site-directed recombination mutant libraries, yielding two recombinant PabUPOs with expression levels of 5.4 and 14.1 mg/liter in Saccharomyces cerevisiae. These variants were subsequently transferred to Pichia pastoris for overproduction in a fed-batch bioreactor, boosting expression levels up to 290 mg/liter, with the highest volumetric activity achieved to date for a recombinant peroxygenase (60,000 U/liter, with veratryl alcohol as the substrate). With a broad pH activity profile, ranging from pH 2.0 to 9.0, these highly secreted, active, and stable peroxygenases are promising tools for future engineering endeavors as well as for their direct application in different industrial and environmental settings. KW - Peroxygenase KW - heterologous functional expression Y1 - 2021 UR - https://journals.asm.org/doi/10.1128/AEM.00878-21 U6 - https://doi.org/10.1128/AEM.00878-21 SN - 1098-5336 SN - 0099-2240 VL - 87 IS - 19 ER - TY - GEN A1 - Babot, Esteban D. A1 - Aranda, Carmen A1 - Kiebist, Jan A1 - Scheibner, Katrin A1 - Ullrich, René A1 - Hofrichter, Martin A1 - Martínez, Angel T. A1 - Gutierrez, Ana T1 - Enzymatic Epoxidation of Long-Chain Terminal Alkenes by Fungal Peroxygenases T2 - Antioxidants N2 - Terminal alkenes are among the most attractive starting materials for the synthesis of epoxides, which are essential and versatile intermediate building blocks for the pharmaceutical, flavoring, and polymer industries. Previous research on alkene epoxidation has focused on the use of several oxidizing agents and/or different enzymes, including cytochrome P450 monooxygenases, as well as microbial whole-cell catalysts that have several drawbacks. Alternatively, we explored the ability of unspecific peroxygenases (UPOs) to selectively epoxidize terminal alkenes. UPOs are attractive biocatalysts because they are robust extracellular enzymes and only require H2O2 as cosubstrate. Here, we show how several UPOs, such as those from Cyclocybe (Agrocybe) aegerita (AaeUPO), Marasmius rotula (MroUPO), Coprinopsis cinerea (rCciUPO), Humicola insolens (rHinUPO), and Daldinia caldariorum (rDcaUPO), are able to catalyze the epoxidation of long-chain terminal alkenes (from C12:1 to C20:1) after an initial optimization of several reaction parameters (cosolvent, cosubstrate, and pH). In addition to terminal epoxides, alkenols and other hydroxylated derivatives of the alkenes were formed. Although all UPOs were able to convert and epoxidize the alkenes, notable differences were observed between them, with rCciUPO being responsible for the highest substrate turnover and MroUPO being the most selective with respect to terminal epoxidation. The potential of peroxygenases for epoxidizing long-chain terminal alkenes represents an interesting and green alternative to the existing synthesis technologies. KW - Peroxygenase KW - oxyfunctionalization KW - epoxidation KW - terminal alkenes KW - epoxides Y1 - 2022 UR - https://www.mdpi.com/2076-3921/11/3/522 U6 - https://doi.org/10.3390/antiox11030522 SN - 2076-3921 VL - 11 IS - 3 SP - 1 EP - 12 ER - TY - GEN A1 - Schramm, Marina A1 - Friedrich, Stephanie A1 - Schmidtke, Kai-Uwe A1 - Panzer, Paul A1 - Kellner, Harald A1 - Ullrich, René A1 - Hofrichter, Martin A1 - Scheibner, Katrin T1 - Cell-Free Protein Synthesis with Fungal Lysates for the Rapid Production of Unspecific Peroxygenases T2 - Antioxidants N2 - Unspecific peroxygenases (UPOs, EC 1.11.2.1) are fungal biocatalysts that have attracted considerable interest for application in chemical syntheses due to their ability to selectively incorporate peroxide-oxygen into non-activated hydrocarbons. However, the number of available and characterized UPOs is limited, as it is difficult to produce these enzymes in homologous or hetero-logous expression systems. In the present study, we introduce a third approach for the expression of UPOs: cell-free protein synthesis using lysates from filamentous fungi. Biomass of Neurospora crassa and Aspergillus niger, respectively, was lysed by French press and tested for translational activity with a luciferase reporter enzyme. The upo1 gene from Cyclocybe (Agrocybe) aegerita (encoding the main peroxygenase, AaeUPO) was cell-free expressed with both lysates, reaching activities of up to 105 U L−1 within 24 h (measured with veratryl alcohol as substrate). The cell-free expressed enzyme (cfAaeUPO) was successfully tested in a substrate screening that included prototypical UPO substrates, as well as several pharmaceuticals. The determined activities and catalytic performance were comparable to that of the wild-type enzyme (wtAaeUPO). The results presented here suggest that cell-free expression could become a valuable tool to gain easier access to the immense pool of putative UPO genes and to expand the spectrum of these sought-after biocatalysts. KW - unspecific peroxygenase KW - monooxygenase KW - cell-free protein synthesis KW - in vitro translation Y1 - 2022 UR - https://www.mdpi.com/2076-3921/11/2/284 U6 - https://doi.org/10.3390/antiox11020284 SN - 2076-3921 VL - 11 IS - 2 SP - 1 EP - 15 ER - TY - GEN A1 - Hofrichter, Martin A1 - Kellner, Harald A1 - Herzog, Robert A1 - Karich, Alexander A1 - Kiebist, Jan A1 - Scheibner, Katrin A1 - Ullrich, René T1 - Peroxide-Mediated Oxygenation of Organic Compounds by Fungal Peroxygenases T2 - Antioxidants N2 - Unspecific peroxygenases (UPOs), whose sequences can be found in the genomes of thousands of filamentous fungi, many yeasts and certain fungus-like protists, are fascinating biocatalysts that transfer peroxide-borne oxygen (from H2O2 or R-OOH) with high efficiency to a wide range of organic substrates, including less or unactivated carbons and heteroatoms. A twice-proline-flanked cysteine (PCP motif) typically ligates the heme that forms the heart of the active site of UPOs and enables various types of relevant oxygenation reactions (hydroxylation, epoxidation, subsequent dealkylations, deacylation, or aromatization) together with less specific one-electron oxidations (e.g., phenoxy radical formation). In consequence, the substrate portfolio of a UPO enzyme always combines prototypical monooxygenase and peroxidase activities. Here, we briefly review nearly 20 years of peroxygenase research, considering basic mechanistic, molecular, phylogenetic, and biotechnological aspects. KW - unspecific peroxygenase KW - monooxygenase KW - peroxidases KW - hydroxylation KW - epoxidation KW - dealkylation Y1 - 2022 UR - https://www.mdpi.com/2076-3921/11/1/163 U6 - https://doi.org/10.3390/antiox11010163 SN - 2076-3921 VL - 11 IS - 1 SP - 1 EP - 21 ER - TY - GEN A1 - König, Rosalie A1 - Kiebist, Jan A1 - Kalmbach, Johannes A1 - Herzog, Robert A1 - Schmidtke, Kai-Uwe A1 - Kellner, Harald A1 - Ullrich, René A1 - Jehmlich, Nico A1 - Hofrichter, Martin A1 - Scheibner, Katrin T1 - Novel unspecific peroxygenase from Truncatella angustata catalyzes the synthesis of bioactive lipid mediators T2 - Microorganisms N2 - Lipid mediators, such as epoxidized or hydroxylated eicosanoids (EETs, HETEs) of arachidonic acid (AA), are important signaling molecules and play diverse roles at different physiological and pathophysiological levels. The EETs and HETEs formed by the cytochrome P450 enzymes are still not fully explored, but show interesting anti-inflammatory properties, which make them attractive as potential therapeutic target or even as therapeutic agents. Conventional methods of chemical synthesis require several steps and complex separation techniques and lead only to low yields. Using the newly discovered unspecific peroxygenase TanUPO from the ascomycetous fungus Truncatella angustata, 90% regioselective conversion of AA to 14,15-EET could be achieved. Selective conversion of AA to 18-HETE, 19-HETE as well as to 11,12-EET and 14,15-EET was also demonstrated with known peroxygenases, i.e., AaeUPO, CraUPO, MroUPO, MweUPO and CglUPO. The metabolites were confirmed by HPLC-ELSD, MS1 and MS2 spectrometry as well as by comparing their analytical data with authentic standards. Protein structure simulations of TanUPO provided insights into its substrate access channel and give an explanation for the selective oxyfunctionalization of AA. The present study expands the scope of UPOs as they can now be used for selective syntheses of AA metabolites that serve as reference material for diagnostics, for structure-function elucidation as well as for therapeutic and pharmacological purposes KW - eicosanoids KW - lipid mediators KW - EETs KW - HETEs KW - unspecific peroxygenases KW - human drug KW - metabolites KW - biocatalysis KW - TanUPO Y1 - 2022 UR - https://www.mdpi.com/2076-2607/10/7/1267 U6 - https://doi.org/10.3390/microorganisms10071267 SN - 2076-2607 VL - 10 IS - 7 SP - 1 EP - 18 ER - TY - GEN A1 - Kellner, Harald A1 - Friedrich, Stephanie A1 - Schmidtke, Kai-Uwe A1 - Ullrich, René A1 - Kiebist, Jan A1 - Zänder, Daniel A1 - Hofrichter, Martin A1 - Scheibner, Katrin T1 - Draft genome sequence of Truncatella angustata (Anamorph) S358 T2 - Microbiology resource announcement N2 - The ascomycete Truncatella angustata has a worldwide distribution. Commonly, it is associated with plants as an endophyte, pathogen, or saprotroph. The genome assembly comprises 44.9 Mbp, a G+C content of 49.2%, and 12,353 predicted genes, among them 12 unspecific peroxygenases (EC 1.11.2.1). KW - unspecific peroxygenase KW - GENOME SEQUENCES KW - Truncatella angustata Y1 - 2022 UR - https://journals.asm.org/doi/epub/10.1128/mra.00052-22 U6 - https://doi.org/10.1128/mra.00052-22 SN - 2169-8287 SN - 2576-098X VL - 11 IS - 7 ER - TY - GEN A1 - Karich, Alexander A1 - Cai, Hongtao A1 - Linhardt, Anne A1 - Antony, Anila A1 - Liers, Christiane A1 - Ullrich, René A1 - Schwaderer, Fabian A1 - Kalmbach, Johannes A1 - Scheibner, Katrin A1 - Hofrichter, Martin A1 - Synytska, Alla T1 - Conductive hairy particles with homogeneous and Janus design as carrier materials for the efficient immobilization of unspecific peroxygenases T2 - Biotechnology journal : systems & synthetic biology, nanobiotech, medicine N2 - Efficient immobilization of unspecific peroxygenases (UPOs) on hairy particles with homogeneous and Janus design possessingconductive core and polymeric shell was demonstrated. PDMAEMA brushes (hairs) with controlled chain lengths weresuccessfully grown from the conductive silver particles allowing further immobilization of enzymes and keeping their activity.The Janus design of the synthesized particles maintained the conductivity of the core material. Enzyme immobilization onbrush-modified particles was first carried out with two laccases as model enzymes [Trametes versicolor (TveLac) and Pycnoporuscinnabarinus (PciLac)] and then successfully extended to UPOs [two wild-type and one recombinant UPO from Marasmius rotula(MroUPO) and Agrocybe aegerita (AaeUPO, rAaeUPO)]. The most efficient immobilization was achieved for MroUPO on Janusparticles. The enzyme loading is reversible and could be successfully repeated after cleaning of the particles. Thus, the successfulimmobilization of an MroUPO on conductive hairy Janus particles was demonstrated for the first time. The major advantage ofthe proposed approach lies in the reusability of the enzyme and its carrier, as well as in the use of conductive core materials, whichcould be promising, for example, as material for electrochemical biosensors in future. KW - Ag particles KW - Core shell particles KW - Enzyme immobilization KW - Janus particles KW - PDMAEMA KW - Polymer brushes KW - Unspecific peroxygenases Y1 - 2025 U6 - https://doi.org/10.1002/biot.70078 SN - 1860-7314 VL - 20 IS - 7 SP - 1 EP - 12 PB - Wiley-VCH CY - Weinheim ER -