TY - GEN A1 - Gröbe, Glenn A1 - Ullrich, René A1 - Pecyna, Marek J. A1 - Kapturska, Danuta A1 - Friedrich, Stephanie A1 - Hofrichter, Martin A1 - Scheibner, Katrin T1 - High-yield production of aromatic peroxygenase by the agaric fungus Marasmius rotula T2 - AMB Express N2 - An extracellular peroxygenase from Marasmius rotula was produced in liquid culture, chromatographically purified and partially characterized. This is the third aromatic peroxygenase (APO) that has been characterized in detail and the first one that can be produced in high yields. The highest enzyme levels of about 41,000 U l-1 (corresponding to appr. 445 mg l-1 APO protein) exceeded the hitherto reported levels more than 40-fold and were detected in carbon- and nitrogen-rich complex media. The enzyme was purified by FPLC to apparent homogeneity (SDS-PAGE) with a molecular mass of 32 kDa (27 kDa after deglycosylation) and isoelectric points between 4.97 and 5.27. The UV-visible spectrum of the native enzyme showed a characteristic maximum (Soret band) at 418 nm that shifted after reduction with sodium dithionite and flushing with carbon monoxide to 443 nm. The pH optimum of the M. rotula enzyme was found to vary between pH 5 and 6 for most reactions studied. The apparent Km-values for 2,6-dimethoxyphenol, benzyl alcohol, veratryl alcohol, naphthalene and H2O2 were 0.133, 0.118, 0.279, 0.791 and 3.14 mM, respectively. M. rotula APO was found to be highly stable in a pH range from 5 to 10 as well as in the presence of organic solvents (50% vol/vol) such as methanol, acetonitrile and N,N-dimethylformamide. Unlike other APOs, the peroxygenase of M. rotula showed neither brominating nor chlorinating activities. KW - Peroxygenase KW - Peroxidase KW - Basidiomycota KW - Cytochrome P450 KW - Bioreactor Y1 - 2011 UR - http://www.amb-express.com/content/1/1/31 SN - 2191-0855 ER - 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 - 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 - 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 - 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 -