TY - GEN A1 - Friedrich, Stephanie A1 - Gröbe, Glenn A1 - Kluge, Martin A1 - Brinkmann, Tobias A1 - Hofrichter, Martin A1 - Scheibner, Katrin T1 - Optimization of a biocatalytic process to gain (R)-1-phenylethanol by applying the software tool Sabento for ecological assessment during the early stages of development T2 - Journal of Molecular Catalysis : B, Enzymatic N2 - Ecological assessment using the software tool Sabento was conducted to compare different processes to gain the fine chemical (R)-1-phenylethanol from ethylbenzene. The software was applied during the biocatalytic process development using the unspecific peroxygenase (EC .11.2.1) of the fungus Agrocybe aegerita. The process could be systematically improved with respect to the ecological performance during process development. Compared to a modern chemical process and a further biotechnological process, it now reaches the best environmental key indicator. The software tool Sabento proved to be well suited to work out the most important factors determining the ecological burdens in the early stages of process development. KW - Ecological assessment KW - Biocatalytic process for (R)-1-phenylethanol KW - Unspecific peroxygenase (EC 1.11.2.1) KW - Agrocybe aegerita Y1 - 2014 UR - http://www.sciencedirect.com/science/article/pii/S1381117713002774 U6 - https://doi.org/10.1016/j.molcatb.2013.10.002 SN - 1381-1177 IS - 103 SP - 36 EP - 40 ER - TY - GEN A1 - Yarman, Aysu A1 - Gröbe, Glenn A1 - Neumann, Bettina A1 - Kinne, Mathias A1 - Gajovic-Eichelmann, Nenad A1 - Wollenberger, Ulla A1 - Hofrichter, Martin A1 - Ullrich, René A1 - Scheibner, Katrin A1 - Scheller, Frieder W. T1 - The aromatic peroxygenase from Marasmius rutola—a new enzyme for biosensor applications T2 - Analytical and Bioanalytical Chemistry N2 - The aromatic peroxygenase (APO; EC 1.11.2.1) from the agraric basidomycete Marasmius rotula (MroAPO) immobilized at the chitosan-capped gold-nanoparticle-modified glassy carbon electrode displayed a pair of redox peaks with a midpoint potential of −278.5 mV vs. AgCl/AgCl (1 M KCl) for the Fe2+/Fe3+ redox couple of the heme-thiolate-containing protein. MroAPO oxidizes aromatic substrates such as aniline, p-aminophenol, hydroquinone, resorcinol, catechol, and paracetamol by means of hydrogen peroxide. The substrate spectrum overlaps with those of cytochrome P450s and plant peroxidases which are relevant in environmental analysis and drug monitoring. In M. rotula peroxygenase-based enzyme electrodes, the signal is generated by the reduction of electrode-active reaction products (e.g., p-benzoquinone and p-quinoneimine) with electro-enzymatic recycling of the analyte. In these enzyme electrodes, the signal reflects the conversion of all substrates thus representing an overall parameter in complex media. The performance of these sensors and their further development are discussed. KW - Unspecific peroxygenase KW - Biosensors KW - Cytochrome P450 KW - Phenolic substances Y1 - 2012 UR - http://link.springer.com/article/10.1007%2Fs00216-011-5497-y U6 - https://doi.org/10.1007/s00216-011-5497-y SN - 1618-2650 VL - 402 IS - 1 SP - 405 EP - 412 ER - TY - GEN A1 - Poraj-Kobielska, Marzena A1 - Atzrodt, Jens A1 - Holla, Wolfgang A1 - Sandvoss, Martin A1 - Gröbe, Glenn A1 - Scheibner, Katrin A1 - Hofrichter, Martin T1 - Preparation of labeled human drug metabolites and drug-drug interaction-probes with fungal peroxygenases T2 - Journal of Labelled Compounds and Radiopharmaceuticals N2 - Enzymatic conversion of a drug can be an efficient alternative for the preparation of a complex metabolite compared with a multi-step chemical synthesis approach. Limitations exist for chemical methods for direct oxygen incorporation into organic molecules often suffering from low yields and unspecific oxidation and also for alternative whole-cell biotransformation processes, which require specific fermentation know-how. Stable oxygen-transferring biocatalysts such as unspecific peroxygenases (UPOs) could be an alternative for the synthesis of human drug metabolites and related stable isotope-labeled analogues. This work shows that UPOs can be used in combination with hydrogen/deuterium exchange for an efficient one-step process for the preparation of 4'-OH-diclofenac-d6. The scope of the reaction was investigated by screening of different peroxygenase subtypes for the transformation of selected deuterium-labeled substrates such as phenacetin-d3 or lidocaine-d3. Experiments with diclofenac-d7 revealed that the deuterium-labeling does not affect the kinetic parameters. By using the latter substrate and H2 (18) O2 as cosubstrate, it was possible to prepare a doubly isotope-labeled metabolite (4'-(18) OH-diclofenac-d6). UPOs offer certain practical advantages compared with P450 enzyme systems in terms of stability and ease of handling. Given these advantages, future work will expand the existing 'monooxygenation toolbox' of different fungal peroxygenases that mimic P450 in vitro reactions. KW - Isotopic labeled synthesis KW - deuterium KW - 4'OH-diclofenac KW - paracetamol KW - phenacetin KW - lidocaine KW - unspecific/aromatic peroxygenase KW - EC 1.11.2.1; KW - human drug metabolites Y1 - 2013 UR - http://onlinelibrary.wiley.com/doi/10.1002/jlcr.3103/abstract;jsessionid=5453A37788468921BE8A03CABF2928DD.f03t02 U6 - https://doi.org/10.1002/jlcr.3103 SN - 1099-1344 VL - 56 IS - 9-10 SP - 513 EP - 519 ER - TY - GEN A1 - Peter, Sebastian A1 - Karich, Alexander A1 - Ullrich, René A1 - Gröbe, Glenn A1 - Scheibner, Katrin A1 - Hofrichter, Martin T1 - Enzymatic one-pot conversion of cyclohexane into cyclohexanone: Comparison of four fungal peroxygenases T2 - Journal of Molecular Catalysis : B, Enzymatic N2 - Unspecific peroxygenases (UPO; EC 1.11.2.1) represent a group of secreted heme-thiolate proteins that are capable of catalyzing the mono-oxygenation of diverse organic compounds, using only H2O2 as a co-substrate. Here we show that the four peroxygenases AaeUPO, MroUPO, rCciUPO and rNOVO catalyze the stepwise hydroxylation of cyclohexane to cyclohexanol and cyclohexanone. The catalytic efficiencies (kcat/Km) for the initial hydroxylation were in the same order of magnitude for all four peroxygenases (∼104 M−1 s−1), whereas they differed in the second step. The conversion of cyclohexanol by AaeUPO and rCciUPO was 1–2 orders of magnitude less efficient (∼102 M−1 s−1) than by MroUPO and rNOVO (∼104 M−1 s−1). The highest conversion rate in terms of H2O2 utilization was accomplished by MroUPO under repeated addition of the peroxide (87% in relation to the total products formed). Using the latter UPO, we successfully established a micro-mixing reaction device (SIMM-V2) for the oxidation of cyclohexane. As cyclohexanone is a chemical of high relevance, for example, as starting material for polymer syntheses or as organic solvent, new enzymatic production pathways for this compound are of interest to complement existing chemical and biotechnological approaches. Stable and versatile peroxygenases, as those presented here, may form a promising biocatalytic platform for the development of such enzyme-based processes. KW - cyclohexane KW - cyclohexanol KW - cyclohexanone KW - UPO KW - Peroxygenase Y1 - 2014 UR - http://www.sciencedirect.com/science/article/pii/S138111771300266X U6 - https://doi.org/10.1016/j.molcatb.2013.09.016 IS - 103 SP - 47 EP - 51 ER - TY - GEN A1 - Piontek, Klaus A1 - Strittmatter, Eric A1 - Ullrich, René A1 - Gröbe, Glenn A1 - Pecyna, Marek J. A1 - Kluge, Martin A1 - Scheibner, Katrin A1 - Hofrichter, Martin A1 - Plattner, Dietmar A. T1 - Structural basis of substrate conversion in a new aromatic peroxygenase: cytochrome P450 functionality with benefits T2 - The Journal of Biological Chemistry N2 - Aromatic peroxygenases (APOs) represent a unique oxidoreductase sub-subclass of heme proteins with peroxygenase and peroxidase activity and were thus recently assigned a distinct EC classification (EC 1.11.2.1). They catalyze, inter alia, oxyfunctionalization reactions of aromatic and aliphatic hydrocarbons with remarkable regio- and stereoselectivities. When compared with cytochrome P450, APOs appear to be the choice enzymes for oxyfunctionalizations in organic synthesis due to their independence from a cellular environment and their greater chemical versatility. Here, the first two crystal structures of a heavily glycosylated fungal aromatic peroxygenase (AaeAPO) are described. They reveal different pH-dependent ligand binding modes. We model the fitting of various substrates in AaeAPO, illustrating the way the enzyme oxygenates polycyclic aromatic hydrocarbons. Spatial restrictions by a phenylalanine pentad in the active-site environment govern substrate specificity in AaeAPO. KW - Cytochrome P450 KW - Fungi; Glycoprotein KW - Oxyfunctionalization KW - Polycyclic Aromatic Hydrocarbons KW - Peroxygenase Y1 - 2013 U6 - https://doi.org/10.1074/jbc.M113.514521 SN - 1083-351X IS - 288 SP - 34767 EP - 34776 ER - TY - GEN A1 - Kluge, Martin A1 - Ullrich, René A1 - Scheibner, Katrin A1 - Hofrichter, Martin T1 - Formation of naphthalene hydrates in the enzymatic conversion of 1,2-dihydronaphthalene by two fungal peroxygenases and subsequent naphthalene formation T2 - Journal of Molecular Catalysis : B, Enzymatic N2 - The formation of naphthalene hydrates (i.e. 1- and 2-hydroxy-1,2-dihydronaphthalene) displays a new activity (besides epoxidation) in the enzymatic transformation of 1,2-dihydronaphthalene by two fungal unspecific peroxygenases (UPOs) accounting for 16–19% of the overall turnover. These arene hydrates decayed into naphthalene that in turn was converted by UPOs into naphthols. The oxygen transferred during hydroxylation was shown to derive from hydrogen peroxide proving a true peroxygenation reaction. KW - Unspecific peroxygenase KW - Naphthalene hydrates KW - Aromatization KW - Oxygenation Y1 - 2014 UR - http://www.sciencedirect.com/science/article/pii/S1381117713002464 U6 - https://doi.org/10.1016/j.molcatb.2013.08.017 SN - 1381-1177 IS - 103 SP - 56 EP - 60 ER - TY - GEN A1 - Kluge, Martin A1 - Ullrich, René A1 - Scheibner, Katrin A1 - Hofrichter, Martin T1 - Stereoselective benzylic hydroxylation of alkylbenzenes and epoxidation of styrene derivatives catalyzed by the peroxygenase of Agrocybe aegerita T2 - Green Chemistry N2 - Here we report on the stereoselective benzylic hydroxylation and C1-C2 epoxidation of alkylbenzenes and styrene derivatives, respectively, by a heme-thiolate peroxygenase (EC 1.11.2.1) from the fungus Agrocybe aegerita. Benzylic hydroxylation led exclusively to the (R)-1-phenylalkanols. For (R)-1-phenylethanol, (R)-1-phenylpropanol and (R)-1-tetralol, the ee reached >99%. For longer chain lengths, the enantiomeric excesses (ee) and total turnover numbers (TTN) decreased while the number of by-products, e.g. 1-phenylketones, increased. Epoxidation of straight chain and cyclic styrene derivatives gave a heterogeneous picture and resulted in moderate to excellent ee values and TTN: e.g., in the case of (1R,2S)-cis-[small beta]-methylstyrene oxide formation, an ee >99% and a TTN of 110 000 was achieved. Hydroxylation and epoxidation were true peroxygenations, which was demonstrated by the incorporation of 18O from H218O2 into the products. The use of fed-batch devices and varying feeding strategies for the substrate and co-substrate turned out to be a suitable approach to optimize peroxygenase catalysis. Y1 - 2012 UR - http://pubs.rsc.org/en/Content/ArticleLanding/2012/GC/C1GC16173C#!divAbstract SN - 1463-9270 IS - 2 SP - 440 EP - 446 ER - TY - GEN A1 - Poraj-Kobielska, Marzena A1 - Kinne, Matthias A1 - Ullrich, René A1 - Scheibner, Katrin A1 - Hofrichter, Martin T1 - A spectrophotometric assay for the detection of fungal peroxygenases T2 - Analytical Biochemistry N2 - Rapid and simple spectrophotometric methods are required for the unambiguous detection of recently discovered fungal peroxygenases in vivo and in vitro. This paper describes a peroxygenase-specific assay using 5-nitro-1,3-benzodioxole as substrate. The product, 4-nitrocatechol, produces a yellow color at pH 7, which can be followed over time at 425 nm (ε425 = 9,700 M−1 cm−1), and a red color when adjusted to pH >12, which can be measured in form of an end-point determination at 514 nm (ε514 = 11,400 M−1 cm−1). The assay is suitable for detecting peroxygenase activities in complex growth media and environmental samples as well as for high-throughput screenings. KW - Monooxygenase KW - Peroxidase KW - Heme-thiolate KW - P450 KW - Chloroperoxidase KW - 5-Nitro-1,3-benzodioxole Y1 - 2012 UR - http://www.sciencedirect.com/science/article/pii/S0003269711006610 UR - 1096-0309 U6 - https://doi.org/10.1016/j.ab.2011.10.009 VL - 421 IS - 1 SP - 327 EP - 329 ER - TY - GEN A1 - Martínez, Angel T. A1 - Ruiz-Dueñas, Francisco J. A1 - Gutiérrez, Ana A1 - Río, José C. del A1 - Alcalde, Miguel A1 - Liers, Christiane A1 - Ullrich, René A1 - Hofrichter, Martin A1 - Scheibner, Katrin A1 - Kalum, Lisbeth A1 - Vind, Jesper A1 - Lund, Henrik T1 - Search, engineering, and applications of new oxidative biocatalysts T2 - Biofuels, Bioproducts and Biorefining N2 - Most industrial enzymes are hydrolases, such as glycosidases and esterases. However, oxidoreductases have an unexploited potential for substituting harsh (and scarcely selective) chemical processes. A group of basidiomycetes are the only organisms degrading the aromatic lignin polymer, enabling the subsequent use of plant polysaccharides. Therefore, these fungi and their ligninolytic peroxidases are the biocatalysts of choice for industrial delignification and oxidative biotransformations of aromatic and other organic compounds. The latter also include oxygenation reactions, which are catalyzed with high regio/stereo selectivity by fungal peroxygenases. In search for novel and more robust peroxidases/peroxygenases, basidiomycetes from unexplored habitats were screened, and hundreds of genes identified in basidiomycete genomes (in collaboration with the DOE JGI). The most interesting genes were heterologously expressed, and the corresponding enzymes structurally-functionally characterized. The information obtained enabled us to improve the enzyme operational and catalytic properties by directed mutagenesis. However, the structural-functional relationships explaining some desirable properties are not established yet and, therefore, their introduction was addressed by ‘non-rational’ directed evolution. Then, over 100 oxidative biotransformations were analyzed. Among them, it is noteworthy to mention the regio/stereo selective hydroxylation of long/short-chain alkanes (a chemically challenging reaction), epoxidation of alkenes, and production of hydroxy-fatty acids. Concerning aromatic oxygenations, the regioselective hydroxylation of flavonoids, and stereoselective hydroxylation/epoxidation of alkyl/alkenyl-benzenes were among the most remarkable reactions, together with enzymatic hydroxylation of benzene (as an alternative for harsh chemical process). Finally, peroxidases and peroxygenases also showed a potential as delignification biocatalysts and in the decolorization of contaminant dyes from textile industries. KW - peroxygenases KW - peroxidases KW - lignin degradation KW - oxidative industrial biocatalysts KW - enzyme rational design KW - directed enzyme evolution KW - selective oxygenation Y1 - 2014 UR - http://onlinelibrary.wiley.com/doi/10.1002/bbb.1498/abstract U6 - https://doi.org/10.1002/bbb.1498 SN - 1932-1031 VL - 8 IS - 6 SP - 819 EP - 835 ER - TY - PAT A1 - Poraj-Kobielska, Marzena A1 - Scheibner, Katrin A1 - Gröbe, Glenn A1 - Kiebist, Jan A1 - Grün, Manfred A1 - Ullrich, René A1 - Hofrichter, Martin T1 - Verfahren zur Deacylierung von Corticoiden Y1 - 2014 ER - TY - GEN A1 - Kluge, Martin A1 - Ullrich, René A1 - Scheibner, Katrin A1 - Hofrichter, Martin T1 - Spectrophotometric assay for detection of aromatic hydroxylation catalyzed by fungal haloperoxidase–peroxygenase T2 - Applied Microbiology and Biotechnology N2 - Agrocybe aegerita peroxidase (AaP) is a versatile heme-thiolate protein that can act as a peroxygenase and catalyzes, among other reactions, the hydroxylation of aromatic rings. This paper reports a rapid and selective spectrophotometric method for directly detecting aromatic hydroxylation by AaP. The weakly activated aromatic compound naphthalene served as the substrate that was regioselectively converted into 1-naphthol in the presence of the co-substrate hydrogen peroxide. Formation of 1-naphthol was followed at 303 nm (ɛ 303 = 2,010 M−1 cm−1), and the apparent Michaelis–Menten (K m) and catalytic (k cat) constants for the reaction were estimated to be 320 μM and 166 s−1, respectively. This method will be useful in screening of fungi and other microorganisms for extracellular peroxygenase activities and in comparing and assessing different catalytic activities of haloperoxidase–peroxygenases. KW - Hydroxylation KW - Peroxygenase Y1 - 2007 UR - http://link.springer.com/article/10.1007%2Fs00253-007-0942-8 U6 - https://doi.org/10.1007/s00253-007-0942-8 SN - 1432-0614 VL - 75 IS - 6 SP - 1473 EP - 1478 ER - 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 - Herzog, Natalie A1 - Hansen, Max A1 - Miethbauer, Sebastian A1 - Schmidtke, Kai-Uwe A1 - Anderer, Ursula A1 - Lupp, Amelie A1 - Sperling, Sebastian A1 - Seehofer, Daniel A1 - Damm, Georg A1 - Scheibner, Katrin A1 - Küpper, Jan-Heiner T1 - Primary-like human hepatocytes genetically engineered to obtain proliferation competence display hepatic differentiation characteristics in monolayer and organotypical spheroid cultures T2 - Cell Biology International N2 - Primary human hepatocytes are in great demand during drug development and in hepatology. However, both scarcity of tissue supply and donor variability of primary cells create a need for the development of alternative hepatocyte systems. By using a lentivirus vector system to transfer coding sequences of Upcyte® proliferation genes, we generated non-transformed stable hepatocyte cultures from human liver tissue samples. Here, we show data on newly generated proliferation-competent HepaFH3 cells investigated as conventional two-dimensional monolayer and as organotypical three-dimensional (3D) spheroid culture. In monolayer culture, HepaFH3 cells show typical cobblestone-like hepatocyte morphology and anchorage-dependent growth for at least 20 passages. Immunofluorescence staining revealed that characteristic hepatocyte marker proteins cytokeratin 8, human serum albumin, and cytochrome P450 (CYP) 3A4 were expressed. Quantitative real-time PCR analyses showed that expression levels of analyzed phase I CYP enzymes were at similar levels compared to those of cultured primary human hepatocytes and considerably higher than in the liver carcinoma cell line HepG2. Additionally, transcripts for phase II liver enzymes and transporter proteins OATP-C, MRP2, Oct1, and BSEP were present in HepaFH3. The cells produced urea and converted model compounds such as testosterone, diclofenac, and 7-OH-coumarin into phases I and II metabolites. Interestingly, phases I and II enzymes were expressed at about the same levels in convenient monolayer cultures and complex 3D spheroids. In conclusion, HepaFH3 cells and related primary-like hepatocyte lines seem to be promising tools for in vitro research of liver functions and as test system in drug development and toxicology analysis. KW - cytochrome P450 enzyme Y1 - 2016 UR - http://onlinelibrary.wiley.com/doi/10.1002/cbin.10574/full U6 - https://doi.org/10.1002/cbin.10574 SN - 1095-8355 VL - 40 IS - 3 SP - 341 EP - 353 ER - TY - GEN A1 - Kiebist, Jan A1 - Holla, Wolfgang A1 - Heidrich, Johannes A1 - Poraj-Kobielska, Marzena A1 - Sandvoss, Martin A1 - Simonis, Reiner A1 - Gröbe, Glenn A1 - Atzrodt, Jens A1 - Hofrichter, Martin A1 - Scheibner, Katrin T1 - One-pot synthesis of human metabolites of SAR548304 by fungal peroxygenases T2 - Bioorganic & Medicinal Chemistry N2 - Unspecific peroxygenases (UPOs, EC 1.11.2.1) have proved to be stable oxygen-transferring biocatalysts for H2O2-dependent transformation of pharmaceuticals. We have applied UPOs in a drug development program and consider the enzymatic approach in parallel to a conventional chemical synthesis of the human metabolites of the bile acid reabsorption inhibitor SAR548304. Chemical preparation of N,N-di-desmethyl metabolite was realized by a seven-step synthesis starting from a late precursor of SAR548304 and included among others palladium catalysis and laborious chromatographic purification with an overall yield of 27%. The enzymatic approach revealed that the UPO of Marasmius rotula is particularly suitable for selective N-dealkylation of the drug and enabled us to prepare both human metabolites via one-pot conversion with an overall yield of 66% N,N-di-desmethyl metabolite and 49% of N-mono-desmethylated compound in two separated kinetic-controlled reactions. KW - Peroxgenase Y1 - 2015 UR - http://www.sciencedirect.com/science/article/pii/S0968089615005295 U6 - https://doi.org/10.1016/j.bmc.2015.06.035 SN - 0968-0896 VL - 23 IS - 15 SP - 4324 EP - 4332 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 - GEN A1 - Olmedo, Andrés A1 - Aranda, Carmen A1 - Rio, José C. del A1 - Kiebist, Jan A1 - Scheibner, Katrin A1 - Martínez, Angel T. A1 - Gutiérrez, Ana T1 - From Alkanes to Carboxylic Acids: Terminal Oxygenation by a Fungal Peroxygenase T2 - Angewandte Chemie International Edition N2 - A new heme–thiolate peroxidase catalyzes the hydroxylation of n-alkanes at the terminal position—a challenging reaction in organic chemistry—with H2O2 as the only cosubstrate. Besides the primary product, 1-dodecanol, the conversion of dodecane yielded dodecanoic, 12-hydroxydodecanoic, and 1,12-dodecanedioic acids, as identified by GC–MS. Dodecanal could be detected only in trace amounts, and 1,12-dodecanediol was not observed, thus suggesting that dodecanoic acid is the branch point between mono- and diterminal hydroxylation. Simultaneously, oxygenation was observed at other hydrocarbon chain positions (preferentially C2 and C11). Similar results were observed in reactions of tetradecane. The pattern of products formed, together with data on the incorporation of 18O from the cosubstrate H218O2, demonstrate that the enzyme acts as a peroxygenase that is able to catalyze a cascade of mono- and diterminal oxidation reactions of long-chain n-alkanes to give carboxylic acids. KW - Peroxyenase Y1 - 2016 UR - http://onlinelibrary.wiley.com/doi/10.1002/anie.201605430/abstract U6 - https://doi.org/10.1002/anie.201605430 SN - 1521-3773 VL - 55 IS - 40 SP - 12248 EP - 12251 ER - TY - GEN A1 - Herzog, Natalie A1 - Hansen, Max A1 - Miethbauer, Sebastian A1 - Schmidtke, Kai-Uwe A1 - Anderer, Ursula A1 - Lupp, Amelie A1 - Sperling, Sebastian A1 - Seehofer, Daniel A1 - Damm, Georg A1 - Scheibner, Katrin A1 - Küpper, Jan-Heiner T1 - Primary like human hepatocytes genetically engineered to obtain proliferation competence display liver biotransformation activity in 2D and 3D culture systems T2 - Cell Biology International Y1 - 2016 SN - 1095-8355 SN - 1065-6995 VL - 40 IS - 3 SP - 341 EP - 353 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 -