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Piontek K.,Albert Ludwigs University of Freiburg | Ullrich R.,Unit of Environmental Biotechnology | Liers C.,Unit of Environmental Biotechnology | Diederichs K.,University of Konstanz | And 2 more authors.
Acta Crystallographica Section F: Structural Biology and Crystallization Communications | Year: 2010

Some litter-decaying fungi secrete haem-thiolate peroxygenases that oxidize numerous organic compounds and therefore have a high potential for applications such as the detoxification of recalcitrant organic waste and chemical synthesis. Like P450 enzymes, they transfer oxygen functionalities to aromatic and aliphatic substrates. However, in contrast to this class of enzymes, they only require H2O2 for activity. Furthermore, they exhibit halogenation activity, as in the well characterized fungal chloroperoxidase, and display ether-cleavage activity. The major form of a highly glycosylated peroxygenase was produced from Agrocybe aegerita culture media, purified to apparent SDS homogeneity and crystallized under three different pH conditions. One crystal form containing two molecules per asymmetric unit was solved at 2.2 Å resolution by SAD using the anomalous signal of the haem iron. Subsequently, two other crystal forms with four molecules per asymmetric unit were determined at 2.3 and 2.6 Å resolution by molecular replacement. © International Union of Crystallography 2010.


Peng L.,University of Potsdam | Wollenberger U.,University of Potsdam | Hofrichter M.,Unit of Environmental Biotechnology | Ullrich R.,Unit of Environmental Biotechnology | And 3 more authors.
Electrochimica Acta | Year: 2010

A biosensor for detecting the aromatic substance 4-nitrophenol based on Agrocybe aegerita peroxygenase (AaP) immobilized with chitosan-stabilized gold nanoparticles is presented here. This biosensor measures the enzymatic product of 4-nitrophenol peroxygenation, 4-nitrocatechol, which is electrochemically detected in the presence of hydrogen peroxide. Cyclic voltammetry and amperometry were used to characterize the proposed biosensor. The linear range of the AaP biosensor for the detection of 4-nitrophenol was between 10 and 30 μM with a detection limit of 0.2 μM (based on the S/N = 3). The catalytic property of AaP to oxidize 4-nitrophenol was compared with two other heme proteins, a camphor-hydroxylating cytochrome P450 monooxygenase (P450 cam, CYP101) and horseradish peroxidase (HRP). The results revealed that only AaP is capable of catalyzing the hydroxylation of 4-nitrophenol into 4-nitrocatechol. Consequently, AaP could be a particularly potent biocatalyst that may fill the gap between cytochrome P450s and common heme peroxidases. © 2010 Elsevier Ltd. All rights reserved.


Kinne M.,Unit of Environmental Biotechnology | Zeisig C.,Unit of Environmental Biotechnology | Ullrich R.,Unit of Environmental Biotechnology | Kayser G.,Unit of Environmental Biotechnology | And 2 more authors.
Biochemical and Biophysical Research Communications | Year: 2010

Fungal peroxygenases have recently been shown to catalyze remarkable oxidation reactions. The present study addresses the mechanism of benzylic oxygenations catalyzed by the extracellular peroxygenase of the agaric basidiomycete Agrocybe aegerita. The peroxygenase oxidized toluene and 4-nitrotoluene via the corresponding alcohols and aldehydes to give benzoic acids. The reactions proceeded stepwise with total conversions of 93% for toluene and 12% for 4-nitrotoluene. Using H2 18O2 as the co-substrate, we show here that H2O2 is the source of the oxygen introduced at each reaction step. A. aegerita peroxygenase resembles cytochromes P450 and heme chloroperoxidase in catalyzing benzylic hydroxylations. © 2010 Elsevier Inc.


Kabiersch G.,University of Helsinki | Rajasarkka J.,University of Helsinki | Ullrich R.,Unit of Environmental Biotechnology | Tuomela M.,University of Helsinki | And 4 more authors.
Chemosphere | Year: 2011

Bisphenol A is an endocrine disrupting compound, which is ubiquitous in the environment due to its wide use in plastic and resin production. Seven day old cultures of the litter-decomposing fungus Stropharia coronilla removed the estrogenic activity of bisphenol A (BPA) rapidly and enduringly. Treatment of BPA with purified neutral manganese peroxidase (MnP) from this fungus also resulted in 100% reduction of estrogenic activity, as analyzed using a bioluminescent yeast assay, and in the formation of polymeric compounds. In cultures of Stropharia rugosoannulata, estrogenic activity also quickly disappeared but temporarily re-emerged in the further course of cultivation. LC-MS analysis of the extracted estrogenic culture liquid revealed [M-H]- ions with m/z values of 219 and 235. We hypothesize that these compounds are ring fission products of BPA, which still exhibit one intact hydroxyphenyl group to interact with estrogen receptors displayed by the yeast. © 2011 Elsevier Ltd.


Beckett R.P.,University of KwaZulu - Natal | Zavarzina A.G.,Moscow State University | Liers C.,Unit of Environmental Biotechnology
Fungal Biology | Year: 2013

Lichens are symbiotic associations of a fungus (usually an Ascomycete) with green algae and/or a cyanobacterium. They dominate on 8% of the world's land surface, mainly in Arctic and Antarctic regions, tundra, high mountain elevations and as components of dryland crusts. In many ecosystems, lichens are the pioneers on the bare rock or soil following disturbance, presumably because of their tolerance to desiccation and high temperature. Lichens have long been recognized as agents of mineral weathering and fine-earth stabilization. Being dominant biomass producers in extreme environments they contribute to primary accumulation of soil organic matter. However, biochemical role of lichens in soil processes is unknown. Our recent research has demonstrated that Peltigeralean lichens contain redox enzymes which in free-living fungi participate in lignocellulose degradation and humification. Thus lichen enzymes may catalyse formation and degradation of soil organic matter, particularly in high-stress communities dominated by lower plants. In the present review we synthesize recently published data on lichen phenol oxidases, peroxidases, and cellulases and discuss their possible roles in lichen physiology and soil organic matter transformations. © 2013 The British Mycological Society.


Liers C.,Unit of Environmental Biotechnology | Ullrich R.,Unit of Environmental Biotechnology | Hofrichter M.,Unit of Environmental Biotechnology | Minibayeva F.V.,Russian Academy of Sciences | Beckett R.P.,University of KwaZulu - Natal
Fungal Genetics and Biology | Year: 2011

Lichens belonging to the order Peltigerales display strong activity of multi-copper oxidases (e.g. tyrosinase) as well as heme-containing peroxidases. The lichen peroxidase was purified to homogeneity from the thallus of Leptogium saturninum (LsaPOX) by fast protein liquid chromatography and then partially characterized. The oligomeric protein occurs as both 79. kDa dimeric and 42. kDa monomeric forms, and displayed broad substrate specificity. In addition to an ability to oxidize classic peroxidase substrates (e.g. 2,6-dimethoxyphenol), the enzyme could convert recalcitrant compounds such as synthetic dyes (e.g. Azure B and Reactive Blue 5), 4-nitrophenol and non-phenolic methoxylated aromatics (e.g. veratryl alcohol). Comparing LsaPOX with a basidiomycete dye-decolorizing (DyP)-type peroxidase from Auricularia auricula-judae showed that the lichen enzyme has a high-redox potential, with oxidation capabilities ranging between those of known plant and fungal peroxidases. Internal peptide fragments show homology (up to 60%) with putative proteins from free-living ascomycetes (e.g. Penicillium marneffei and Neosartorya fischeri), but not to sequences of algal or cyanobacterial peptides or to known fungal, bacterial or plant peroxidases. LsaPOX is the first heme peroxidase purified from an ascomyceteous lichen that may help the organism to successfully exploit the extreme micro-environments in which they often grow. © 2011 Elsevier Inc.


Beckett R.P.,University of KwaZulu - Natal | Minibayeva F.V.,Russian Academy of Sciences | Liers C.,Unit of Environmental Biotechnology
Lichenologist | Year: 2013

Abstract In our earlier work, we demonstrated that the oxidases tyrosinase and laccase occur widely in lichens from the Peltigerales. Recently, we discovered the occurrence of another oxidoreductase, a heme peroxidase, in the Peltigeralean 'jelly lichens' Leptogium and Collema. Here we present the results of a survey of peroxidase activity in a range of lichens. In addition to the jelly lichens, strong peroxidase activity also occurs within the Peltigeralean genera Lobaria, Pseudocyphellaria and Sticta. Significant activity occurs in the cell wall, and, unlike laccase activity, peroxidase activity increases considerably following the rehydration of dry thalli. However, activity is absent from Peltigera and from the non-Peltigeralean species tested here. Electrophoretic investigation showed that lichen peroxidases are oligomeric. Possible roles for peroxidases in lichen biology are discussed. Copyright © British Lichen Society 2013.


Gutierrez A.,CSIC - Institute of Natural Resources and Agriculture Biology of Seville | Babot E.D.,CSIC - Institute of Natural Resources and Agriculture Biology of Seville | Ullrich R.,Unit of Environmental Biotechnology | Hofrichter M.,Unit of Environmental Biotechnology | And 2 more authors.
Archives of Biochemistry and Biophysics | Year: 2011

Reaction of fatty acids, fatty alcohols, alkanes, sterols, sterol esters and triglycerides with the so-called aromatic peroxygenase from Agrocybe aegerita was investigated using GC-MS. Regioselective hydroxylation of C 12-C20 saturated/unsaturated fatty acids was observed at the ω-1 and ω-2 positions (except myristoleic acid only forming the ω-2 derivative). Minor hydroxylation at ω and ω-3 to ω-5 positions was also observed. Further oxidized products were detected, including keto, dihydroxylated, keto-hydroxy and dicarboxylic fatty acids. Fatty alcohols also yielded hydroxy or keto derivatives of the corresponding fatty acid. Finally, alkanes gave, in addition to alcohols at positions 2 or 3, dihydroxylated derivatives at both sides of the molecule; and sterols showed side-chain hydroxylation. No derivatives were found for fatty acids esterified with sterols or forming triglycerides, but methyl esters were ω-1 or ω-2 hydroxylated. Reactions using H2 18O2 established that peroxide is the source of the oxygen introduced in aliphatic hydroxylations. These studies also indicated that oxidation of alcohols to carbonyl and carboxyl groups is produced by successive hydroxylations combined with one dehydration step. We conclude that the A. aegerita peroxygenase not only oxidizes aromatic compounds but also catalyzes the stepwise oxidation of aliphatic compounds by hydrogen peroxide, with different hydroxylated intermediates. © 2011 Elsevier Inc. All rights reserved.


Huang Z.,University of Wyoming | Liers C.,Unit of Environmental Biotechnology | Ullrich R.,Unit of Environmental Biotechnology | Hofrichter M.,Unit of Environmental Biotechnology | Urynowicz M.A.,University of Wyoming
Fuel | Year: 2013

This study evaluated the influence of four chemical pretreatment agents (HNO3, catalyzed H2O2, KMnO4, NaOH) on the subsequent enzymatic conversion of subbituminous coal by a fungal manganese peroxidase (MnP) produced by the agaric white-rot fungus Bjerkandera adusta. The effects of the combined chemical and enzymatic treatments were analyzed by high performance size exclusion chromatography (HPSEC) and 3-dimensional excitation emission matrix fluorescence spectroscopy (3D-EEM). The nature of pretreatment agents and their applied concentrations had significant impacts on subsequent enzymatic conversion of coal. The 3D-EEM spectroscopic analysis provided new insight into the nature of the depolymerized and released coal constituents. Using the fluorescence spectra, it was possible to distinguish among humic-like, fulvic acid-like, protein-like, and aromatic/PAH-like substances. The fungal enzyme MnP had little effect on the untreated coal controls. Nitric acid (HNO3) was the most effective pretreatment agent as indicated by the HPSEC profiles, followed by catalyzed H2O2 and KMnO4. Low molecular weight aromatic fragments with sizes ranging from 1.1 to 6.2 kDa were released by all of the pretreatment agents used in combination with MnP. For KMnO4 and HNO3 pretreated coal, all four EEM regions increased after MnP treatment (for example, 307.5/422 nm and 232.5/426 nm, and 340/448 nm and 242.5/484 nm for humic and fulvic acid-like fragments, respectively). © 2013 Elsevier Ltd. All rights reserved.


Liers C.,Unit of Environmental Biotechnology | Bobeth C.,Unit of Environmental Biotechnology | Pecyna M.,Unit of Environmental Biotechnology | Ullrich R.,Unit of Environmental Biotechnology | Hofrichter M.,Unit of Environmental Biotechnology
Applied Microbiology and Biotechnology | Year: 2010

The jelly fungus Auricularia auricula-judae produced an enzyme with manganese-independent peroxidase activity during growth on beech wood (∼300 U l-1). The same enzymatic activity was detected and produced at larger scale in agitated cultures comprising of liquid, plant-based media (e.g. tomato juice suspensions) at levels up to 8,000 U l-1. Two pure peroxidase forms (A. auricula-judae peroxidase (AjP I and AjP II) could be obtained from respective culture liquids by three chromatographic steps. Spectroscopic and electrophoretic analyses of the purified proteins revealed their heme and peroxidase nature. The N-terminal amino acid sequence of AjP matched well with sequences of fungal enzymes known as "dye-decolorizing peroxidases". Homology was found to the N-termini of peroxidases from Marasmius scorodonius (up to 86%), Thanatephorus cucumeris (60%), and Termitomyces albuminosus (60%). Both enzyme forms catalyzed not only the conversion of typical peroxidase substrates such as 2,6-dimethoxyphenol and 2,2′-azino-bis(3-ethylthiazoline-6-sulfonate) but also the decolorization of the high-redox potential dyes Reactive Blue 5 and Reactive Black 5, whereas manganese(II) ions (Mn2+) were not oxidized. Most remarkable, however, is the finding that both AjPs oxidized nonphenolic lignin model compounds (veratryl alcohol; adlerol, a nonphenolic β-O-4 lignin model dimer) at low pH (maximum activity at pH 1.4), which indicates a certain ligninolytic activity of dye-decolorizing peroxidases. © 2009 Springer-Verlag.

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