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Deadwood decay is an important ecosystem process in forest ecosystems, but the relative contribution of specific wood properties of tree species, activities of wood-degrading enzymes, and decomposer communities such as fungi and insects is unclear. We ask whether wood properties, in particular differences between angiosperms and gymnosperms, and organismic diversity of colonizers contribute to wood decomposition. To test this, we exposed deadwood logs of 13 tree species, covering four gymnosperms and nine angiosperm species, in 30 plots under different forest management in three regions in Germany. After a decomposition time of 6.5 years Carpinus betulus and Fagus sylvatica showed the highest decay rates. We found a positive correlation of decay rate with enzyme activities, chemical wood properties (S, K concentration) and organismic diversity, while, heartwood character, lignin content, extractive concentration and phenol content were negatively correlated with decay rate across all 13 tree species. By applying a multi-model inference approach we found that the activity of the wood-degrading enzymes laccase and endocellulase, beetle diversity, heartwood presence, wood ray height and fungal diversity were the most important predictor variables for wood decay. Although we were not able to identify direct cause and effect relations by our approach, we conclude that enzyme activity and organismic diversity are the main drivers of wood decay rate, which greatly differed among tree species. Maintaining high tree species diversity will therefore result in high structural deadwood diversity in terms of decay rate and decay stage.

Three genes have been identified through sequence analysis to encode putative AtzD/TrzD and AtzE enzymes and a TrzR transcriptional regulator via deduced amino acid sequences of functional AtzD/TrzD and/or AtzE published in the literature via a query sequence for homologues at the protein level using BLASTP. The operon was predicted to encode an s-triazine ring-opening amidohydrolase, TrzD (FraEuI1c_3137) and a GntR family transcriptional regulator, TrzR (FraEuI1c_3136), which may regulate the expression of ring-cleavage enzyme whereas the putative atzE (FraEuI1c_1007) gene encodes aspartyl/glutamyl-tRNA (Asn/Gln) amidotransferase subunit A in the course of s-triazine degradation by Frankia strain EuI1c. LC-MS analysis of Frankia sp EuI1c culture filtrates grown in the presence of atrazine or desethyl-desisopropylatrazine revealed a metabolite with a molecular ion (major peak) of m/z 102.7, which was identified as biuret. The trzD (FraEuI1c_3137) gene expression increased up to 4.7-fold in its abundance under 2 mM atrazine exposure when qRT-PCR was applied. Moreover, the mRNA level of the putative trzR (FraEuI1c_3136) gene that is proposed to regulate the gene function of FraEuI1c_3137 exhibited a dose-response and peaked at 2 mM atrazine with a 6.5 fold-increased mRNA level. The putative atzE (FraEuI1c_1007) mRNA level exhibited dose-respond and upregulated up to 10-fold change under the same stress dose.

Enantiomerically pure α-substituted β-hydroxyesters are important chiral building blocks for ligands, auxiliaries and β-lactam antibiotics. A two-step chemo-enzymatic procedure using lipase as biocatalyst is an efficient way to synthesize such products. To date, the methods described are limited to molecules that do not contain a chiral center adjacent to the racemic carbinol, and furthermore, they are limited to acylation. Here, we investigated the deracemization of diastereomerically pure syn- and anti-α-substituted β-hydroxyesters containing two stereo centers, using experimental methods under neat conditions and classical molecule dynamics (MD) simulation. A screening of free and immobilized commercial lipases identified immobilized lipase B from Candida antarctica (Novozyme 435) as the most appropriate biocatalyst for sterically demanding α-substituted β-hydroxyesters. Using Novozyme 435, reaction conditions were optimized and hydroxyesters (3S) or (3R) were achieved with enantiomeric excesses up to ≥99% ee and maximum overall yields of 80%. The absolute configuration of the enantiomers was eventually determined by 1H-NMR spectroscopy after derivatization with Mosher's reagent (α-methoxy-α-trifluoromethylphenylacetic acid = MTPA).

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.

In forest ecosystems, deadwood is an important component that provides habitat and contributes to nutrient cycles, as well as to carbon and water storage. The change of wood constituents, nutrients and microbial species richness in the field over the whole time of decomposition has only rarely been studied, in particular not in relation to oxidative enzyme activities (mediating lignin degradation) and different forest management regimes. To describe wood decomposition, we selected coarse woody debris (CWD) in form of 197 logs of Fagus sylvatica, Picea abies and Pinus sylvestris in forests with different management regimes across three regions in Germany. They were sampled and analyzed for wood density, water content, wood constituents (Klason and acid-soluble lignin, organic extractives, water-soluble lignin fragments), carbon, nitrogen and metals (Al, Ca, Cu, K, Mg, Mn and Zn). Furthermore, the activities of oxidative enzymes like laccase, manganese peroxidase, and general peroxidase were measured. Since filamentous fungi (Basidiomycota, Ascomycota) are the major biological agents of wood decomposition, fungal species richness based on sporocarps and molecular fingerprints was recorded. Higher forest management intensity had a negative effect on deadwood volume and in consequence on fungal species richness (sporocarps), but hardly to other analyzed variables. Furthermore, there were significant differences between the tree species for the concentrations of wood constituents and most nutrients as well as the activities of oxidative enzymes, although their course during decomposition was mostly similar among the tree species. We found that molecular species richness increased with the period of decomposition in contrast to the number of fruiting species, which was highest in the intermediate stage of decomposition. Both types of species richness increased with increasing volume of the CWD logs. Regarding the entire period of decomposition, white-rot fungi (WRF), based on identification of sporocarps, were the most abundant group of wood-decaying fungi in all three tree species. This corresponds well with the overall presence of laccase and peroxidases and the concomitant substantial loss of lignin, which points to the importance of these enzymes in deadwood decomposition. We found a continuous decomposition and decline of volume-related concentrations in wood constituents and nutrients with time of decomposition. Contrary to volume-related concentrations, the concentrations related to dry mass frequently increased.

Decomposition rates of coarse woody debris (CWD) have been investigated in many studies, however data on fungal biomass and the related enzymatic activities in decomposing CWD are scarce. Here, we investigated the relations between fungal biomass, enzyme activities and CWD properties in sap- and heartwood of decomposing logs of 13 different temperate European tree species, exposed for 6 years on the ground. Fungal biomass was significantly higher in sapwood than in heartwood and higher in deciduous than in coniferous species, and represented 0.3–4.4% of CWD dry mass. In deciduous sapwood, fungal biomass may represent up to 29% of the total N stock in CWD. Fungal biomass correlated positively with the N content of CWD and, in heartwood, negatively with extractives. Enzyme activities were higher in deciduous than in coniferous CWD and for hydrolases higher in sapwood than in heartwood. Correlations between enzyme activities and the ergosterol content were generally weak. Hydrolytic enzymes were frequently found in all decaying tree species, whereas ligninolytic oxidoreductases showed high variability specifically in deciduous wood. Fungal biomass and enzymatic activities confirm the assumption that, in the initial stage, the decomposition of deciduous CWD is faster than of coniferous CWD under comparable conditions.

Wood-inhabiting fungi are major agents of wood decomposition. However, it is unclear which factors determine their distribution and enzyme production. Many studies that have addressed this issue suffer from a lack of geographic extent. Here, we investigate the fungal community structure of 117 Fagus sylvatica logs in relation to wood physico-chemical properties and secreted ligninolytic enzymes, across three distinct geographical regions of Germany. Our results revealed that fungal community structure was similar across different regions, but was nevertheless variable in all regions. The relationships between fungal community structure, wood physico-chemical properties and enzyme activities were not consistent across different regions. However, we identified that the wood physico-chemical properties (i.e. decay class, remaining mass, density, extractives, total lignin and pH) were the most important factors associated with the fungal community structure in all three regions. In contrast, the wood physico-chemical properties and the fungal community structure did not sufficiently explain variation in the detected enzymatic activities. Thus, we assume that interspecific interactions and recently described priority effects play more important roles in the production of lignin modifying enzymes.

The ability of Trametes versicolor strain BAFC 2234 to degrade4-nitrophenol in vivo and in vitro was evaluated. T. versicolor grew inthe presence of 0.5 mM 4-nitrophenol and degraded 98.4% of this toxiccompound in less than 96 h. The strain secreted different ligninolyticoxidoreductases such as laccase, Mn-peroxidase and versatile peroxidase.Substantial conversion of nitrophenol, a typical high-redox potentialphenolic substrate, is reported for versatile peroxidase for the firsttime; 2,4-dinitrophenol and a dimer were identified as products. T.versicolor immobilized on natural sponge-like material from Luffaaegyptiaca removed 97% of 4-nitrophenol (1 mM) over a period of 72 h. 4-Nitrophenol phytotoxicity decreased noticeably after fungal treatment. Fil: Levin, Laura Noemi. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Micología y Botánica. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Micología y Botánica; Argentina Fil: Carabajal, Maira Lia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Micología y Botánica. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Micología y Botánica; Argentina Fil: Hofrichter, Martin. Technical University of Dresden; Alemania Fil: Ullrich, René. Technical University of Dresden; Alemania

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.

Substantial conversion of nitrophenols, typical high-redox potential phenolic substrates, by heme peroxidases has only been reported for lignin peroxidase (LiP) so far. But also a dye-decolorizing peroxidase of Auricularia auricula-judae (AauDyP) was found to be capable of acting on (i) ortho-nitrophenol (oNP), (ii) meta-nitrophenol (mNP) and (iii) para-nitrophenol (pNP). The pH dependency for pNP oxidation showed an optimum at pH 4.5, which is typical for phenol conversion by DyPs and other heme peroxidases. In the case of oNP and pNP conversion, dinitrophenols (2,4-DNP and 2,6-DNP) were identified as products and for pNP additionally p-benzoquinone. Moreover, indications were found for the formation of random polymerization products originating from initially formed phenoxy radical intermediates. Nitration was examined using (15)N-labeled pNP and Na(14)NO2 as an additional source of nitro-groups. Products were identified by HPLC-MS, and mass-to-charge ratios were evaluated to clarify the origin of nitro-groups. The additional nitrogen in DNPs formed during enzymatic conversion was found to originate both from (15)N-pNP and (14)NO2Na. Based on these results, a hypothetical reaction scheme and a catalytically responsible confine of the enzyme's active site are postulated.

An efficient heterologous expression system for Auricularia auricula-judae dye-decolorizing peroxidase (DyP) has been constructed. DNA coding for the mature protein sequence was cloned into the pET23a vector and expressed in Escherichia coli BL21(DE3)pLysS. Recombinant DyP was obtained in high yield as inclusion bodies, and different parameters for its in vitro activation were optimized with a refolding yield of ∼8.5% of the E. coli-expressed DyP. Then, a single chromatographic step allowed the recovery of 17% of the refolded DyP as pure enzyme (1.5 mg per liter of culture). The thermal stabilities of wild DyP from A. auricula-judae and recombinant DyP from E. coli expression were similar up to 60 °C, but the former was more stable in the 62–70 °C range. Stabilities against pH and H2O2 were also measured, and a remarkably high stability at extreme pH values (from pH 2 to 12) was observed. The kinetic constants of recombinant DyP for the oxidation of different substrates were determined and, when compared with those of wild DyP, no important differences were ascertained. Both enzymes showed high affinity for Reactive Blue 19 (anthraquinone dye), Reactive Black 5 (azo dye), 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) and 2,6-dimethoxyphenol, with similar acidic pH optima and oxidative stabilities. Oxidation of veratryl alcohol and a nonphenolic lignin model dimer were confirmed, although as minor enzymatic activities. Interestingly, two sets of kinetic constants could be obtained for the oxidation of Reactive Blue 19 and other substrates, suggesting the existence of more than one oxidation site in this new peroxidase family.

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 1.11.2.1) 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.

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.

Over the last years, novel peroxidases secreted by lignocellulose-degrading agaricomycetes have been discovered. Among them, the so-called DyP-type peroxidases (DyPs) that are secreted under conditions close to nature (i.e. in wood cultures) are of particular interest, since they are able to oxidize diverse substrates including veratryl alcohol, non-phenolic lignin model dimers as well as recalcitrant phenols and dyes. In spite of their unique protein structure and their catalytic versatility, the estimation of the redox potential of this new peroxidase group is still pending. To solve this problem, we used a catalytic approach developed by Ayala et al., 2007 [21] , which is based on the Marcus equation and the determination of the redox thermodynamics between heme-peroxidase compound II and the resting state enzyme. Five fungal DyPs (among them four wild-type enzymes and one recombinant protein) were tested regarding phenol oxidation in comparison to other well-studied plant and fungal peroxidases (soybean peroxidase, SBP, Coprinopsis cinerea peroxidase, CiP, lignin peroxidase of Phanerochaete chrysosporium , LiP). DyP-type peroxidases have a high affinity for phenols and can oxidize even recalcitrant representatives such as p -nitrophenol. Based on this “phenol oxidation method”, their redox potential was estimated to range between 1.10 ± 0.02 and 1.20 ± 0.1 V, which is between the values calculated for high-redox potential LiP (1.26 ± 0.17 V) and low-redox potential, phenol-oxidizing plant (0.93 ± 0.04 V for SBP) and fungal (1.06 ± 0.07 V for CiP) peroxidases.

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 H 2 O 2 as a co-substrate. Here we show that the four peroxygenases Aae UPO, Mro UPO, r Cci UPO and rNOVO catalyze the stepwise hydroxylation of cyclohexane to cyclohexanol and cyclohexanone. The catalytic efficiencies ( k cat / K m ) for the initial hydroxylation were in the same order of magnitude for all four peroxygenases (∼10 4 M −1 s −1 ), whereas they differed in the second step. The conversion of cyclohexanol by Aae UPO and r Cci UPO was 1–2 orders of magnitude less efficient (∼10 2 M −1 s −1 ) than by Mro UPO and rNOVO (∼10 4 M −1 s −1 ). The highest conversion rate in terms of H 2 O 2 utilization was accomplished by Mro UPO 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.

The enzymatic oxyfunctionalization of organic molecules under physiological conditions has attracted keen interest from the chemical community. Unspecific peroxygenases (EC 1.11.2.1) secreted by fungi represent an intriguing enzyme type that selectively transfers peroxide-borne oxygen with high efficiency to diverse substrates including unactivated hydrocarbons. They are glycosylated heme-thiolate enzymes that form a separate superfamily of heme proteins. Among the catalyzed reactions are hydroxylations, epoxidations, dealkylations, oxidations of organic hetero atoms and inorganic halides as well as one-electron oxidations. The substrate spectrum of fungal peroxygenases and the product patterns show similarities both to cytochrome P450 monooxygenases and classic heme peroxidases. Given that selective oxyfunctionalizations are among the most difficult to realize chemical reactions and that respectively transformed molecules are of general importance in organic and pharmaceutical syntheses, it will be worth developing peroxygenase biocatalysts for industrial applications.

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.

This study evaluated the influence of four chemical pretreatment agents (HNO 3 , catalyzed H 2 O 2 , KMnO 4 , 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 (HNO 3 ) was the most effective pretreatment agent as indicated by the HPSEC profiles, followed by catalyzed H 2 O 2 and KMnO 4 . 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 KMnO 4 and HNO 3 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).

Dye-decolorizing peroxidases (DyPs) belong to the large group of heme peroxidases. They utilize hydrogen peroxide to catalyze oxidations of various organic compounds. AauDyPI from Auricularia auricula-judae (fungi) was crystallized, and its crystal structure was determined at 2.1 A resolution. The mostly helical structure also shows a β-sheet motif typical for DyPs and Cld (chlorite dismutase)-related structures and includes the complete polypeptide chain. At the distal side of the heme molecule, a flexible aspartate residue (Asp-168) plays a key role in catalysis. It guides incoming hydrogen peroxide toward the heme iron and mediates proton rearrangement in the process of Compound I formation. Afterward, its side chain changes its conformation, now pointing toward the protein backbone. We propose an extended functionality of Asp-168, which acts like a gatekeeper by altering the width of the heme cavity access channel. Chemical modifications of potentially redox-active amino acids show that a tyrosine is involved in substrate interaction. Using spin-trapping experiments, a transient radical on the surface-exposed Tyr-337 was identified as the oxidation site for bulky substrates. A possible long-range electron transfer pathway from the surface of the enzyme to the redox cofactor (heme) is discussed.

A novel sampling method to evaluate extracellular fungal enzyme activities was developed and the validity tested for agar media. The method is based on centrifugation of small agar pieces taken from growing fungal solid-state cultures. Centrifuge tubes that allow spinning liquid out from small samples containing, for example, the hyphal front of a growing mycelium are essential for the protocol. Centrifugation recovers a liquid phase from the samples, which contains soluble material including many enzymes. The recovery of two added model enzymes, namely laccase and manganese peroxidase (MnP), from agar media was sufficient (ranging from 50 % to 75 %) but the addition of humic material into agar decreased the observed MnP activity significantly to approx. 25 % of the stock solution. Using growing cultures, the presence of humus as well as Scots pine sawdust on Hagem’s agar plates induced the production of laccase and peroxidase in certain fungi, which indicates that the method is suitable for screening enzyme activities on different growth media or with variable additives or growth conditions. The use of the presented sampling method for functional enzyme fingerprinting of different fungi may be a promising tool for investigating the behaviour and ecological role of forest soil fungi. This method also allows obtaining spatial data from very small and defined areas of solid fungal cultures, e.g. from microcosms.

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.

Aromatic peroxygenases are extracellular fungal biocatalysts that selectively oxidize a variety of organic compounds. We found that the peroxygenase of the fungus Agrocybe aegerita (AaeAPO) catalyzes the H2O2-dependent hydroxylation of diverse flavonoids. The reactions proceeded rapidly and regioselectively yielding preferentially monohydroxylated products, e.g., from flavanone, apigenin, luteolin, flavone as well as daidzein, quercetin, kaempferol, and genistein. In addition to hydroxylation, O-demethylation of fully methoxylated tangeretin was catalyzed by AaeAPO. The enzyme was merely lacking activity on the quercetin glycoside rutin, maybe due to sterical hindrance by the bulky sugar substituents. Mechanistic studies indicated the presence of epoxide intermediates during hydroxylation and incorporation of H2O2-derived oxygen into the reaction products. Our results raise the possibility that fungal peroxygenases may be useful for versatile, cost-effective, and scalable syntheses of flavonoid metabolites.

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.

Litter decomposing basidiomycetous fungi produce ligninolytic oxidases and peroxidases which are involved in the transformation of lignin, as well as humic and fulvic acids. The aim of this work was to evaluate their importance in lignin transformation in forest litter. Two litter decomposing basidiomycete species differing in their abilities to degrade lignin – Hypholoma fasciculare, and Gymnopus erythropus – were cultured on sterile or non-sterile oak litter and their transformation of a 14C-labelled synthetic lignin (dehydrogenation polymer 14C-DHP) was compared with that of the indigenous litter microflora. Both in sterile and non-sterile litter, colonisation by basidiomycetes led to higher titres of lignocellulose-degrading enzymes, in particular of laccase and Mn-peroxidase (MnP). The titres of the latter were 6 to 40-fold increased in the presence of basidiomycetes compared to non-sterile litter. During 10 weeks, G. erythropus mineralised over 31% of 14C-DHP in sterile litter and 23% in non-sterile litter compared to 14% in the non-sterile control. Lignin mineralization by H. fasciculare was comparable to the non-sterile control, 12% in sterile litter and 16% in the non-sterile litter. The largest part of 14C from 14C-DHP was transformed into humic compounds during litter treatment with both fungi as well as in the control. In addition to the fast lignin mineralization, microcosms containing G. erythropus also showed a lower final content of unaltered lignin and 23–28% of the lignin was converted into water-soluble compounds with relatively low molecular mass (

The extracellular heme-thiolate peroxygenase of Agrocybe aegerita catalyzed the H2O2-dependent hydroxylation of 2-phenoxypropionic acid (POPA) to give the herbicide precursor 2-(4-hydroxyphenoxy)propionic acid (HPOPA). The reaction proceeded regioselectively with an isomeric purity near 98%, and yielded the desired R-isomer of HPOPA with an enantiomeric excess of 60%. 18O-labeling experiments showed that the phenolic hydroxyl in HPOPA originated from H2O2, which establishes that the reaction is mechanistically a peroxygenation. Our results raise the possibility that fungal peroxygenases may be useful for a variety of organic oxidations.

This study deals with the laccase-catalyzed decolorization of azo and anthraquinone dyes. Both purified laccase (Lacc I and Lacc II) as well as the crude enzyme from the white-rot fungus Cerrena unicolor were used to convert the dyes at pH 3.5 (optimum of laccase activity) in aqueous solution. Biotransformation of the dyes was followed spectrophotometrically and confirmed by high performance liquid chromatography (HPLC). The results indicate that the decolorization mechanism follows Michaelis–Menten kinetic and that the initial rate of decolorization depends both on the structure of the dye and on the dye concentration. The saturation constants ( K m ) of purified laccase isoforms (Lacc I, II) differ to some extend indicating different substrate affinities. Surprisingly, one recalcitrant azo dye (AR 27) was decolorized merely by purified laccase in the absence of any redox mediator.

The hard wood-colonizing ascomycete Xylaria polymorpha , that is seemingly lacking peroxidases, produces laccase as sole ligninolytic oxidoreductase. The fungus secreted the enzyme preferably during the growth in complex media based on tomato juice. Addition of 2,5-xylidine considerably stimulated laccase production (up to 14,000 U l −1 ). The enzyme was purified to homogeneity by anion exchange and size exclusion chromatography and characterized by biochemical and molecular methods. Xylaria laccase has a molecular mass of 67 kDa, a p I of 3.1 and an absorption maximum at 605 nm that is characteristic for blue copper proteins. It oxidized all typical laccase substrates including ABTS, 2,6-dimethoxyphenol, guaiacol as well as syringaldazine (catalytic efficiencies 3 × 10 3 to 7 × 10 4 M −1 s −1 ). The deduced amino acid sequence of one amplified laccase gene sequence between the copper binding regions 1 and 3 showed a high level of identity to some other laccases from ascomycetes. Furthermore, the sequence of an internal peptide fragment of the purified laccase was identical with an amino acid sequence deduced from the nucleotide sequence of the laccase gene. Xylaria laccase was found to oxidize a non-phenolic β- O -4 lignin model compound in presence of 1-hydroxybenzotriazole into the corresponding keto-form. The results of this study show that – in addition to ligninolytic basidiomycetes – also wood-dwelling ascomycetes can produce high titers of laccase that may be involved in the oxidation of lignin.

Due to the production of lignocellulose-degrading enzymes, saprotrophic basidiomycetes can significantly contribute to the turnover of soil organic matter. The production of lignin- and polysaccharide-degrading enzymes and changes of the chemical composition of litter were studied with three isolates from a Quercus petraea forest. These isolates were capable of fresh litter degradation and were identified as Gymnopus sp., Hypholoma fasciculare and Rhodocollybia butyracea. Within 12 weeks of incubation, H. fasciculare decomposed 23%, R. butyracea 32% and Gymnopus sp. 38% of the substrate dry mass. All fungi produced laccase and Mn-peroxidase (MnP) and none of them produced lignin peroxidase or other Mn-independent peroxidases. There was a clear distinction in the enzyme production pattern between R. butyracea or H. fasciculare compared to Gymnopus sp. The two former species caused the fastest mass loss during the initial phase of litter degradation, accompanied by the temporary production of laccase (and MnP in H. fasciculare) and also high production of hydrolytic enzymes that later decreased. In contrast, Gymnopus sp. showed a stable rate of litter mass loss over the whole incubation period with a later onset of ligninolytic enzyme production and a longer lasting production of both lignin and cellulose-degrading enzymes. The activity of endo-cleaving polysaccharide hydrolases in this fungus was relatively low but it produced the most cellobiose hydrolase. All fungi decreased the C/N ratio of the litter from 24 to 15–19 and Gymnopus sp. also caused a substantial decrease in the lignin content. Analytical pyrolysis mass spectrometry of litter decomposed by this fungus showed changes in the litter composition similar to those caused by white-rot fungi during wood decay. These changes were less pronounced in the case of H. fasciculare and R. butyracea. All fungi also changed the mean masses of humic acid and fulvic acid fractions isolated from degraded litter. The humic acid fraction after degradation by all three fungi contained more lignin and less carbohydrates. Compared to the decomposition by saprotrophic basidiomycetes, litter degradation in situ on the site of fungal isolation resulted in the relative enrichment of lignin and differences in lignin composition revealed by analytical pyrolysis. It can most probably be explained by the participation of non-basidiomycetous fungi and bacteria during natural litter decomposition.

This paper describes for the first time in detail the lignocellulose degradation system in Paecilomyces inflatus. The fungal genus Paecilomyces contributes the carbon turnover from lignin and carbohydrate plant residues, particularly in compost and soil environment, where basidiomycetes appear very seldom. We studied three different strains of P. inflatus, obtained from different ecophysiological and geographical origin. Various cultivation conditions were employed, and the chemical analysis of decayed straw, compost, birch and spruce wood chips indicated variable responses. Endoglucanase, xylanase and laccase were assayed. All strains of P. inflatus, regardless of their origin, altered the ambient pH in a similar manner in all investigated substrates, suggesting that all P. inflatus isolates may share the common regulatory system to control their environmental pH. The variability among strains of P. inflatus in their ability to remove lignocellulose components often was related to the nature of the substrate and the production of specific enzymes although it was not strictly correlated. This may implicate that other enzymes and/or even other parameters needed for lignocellulosics degradation in P. inflatus should be evaluated. Indications for specific adaptation strategies that may operate in P. inflatus were found.

Manganese peroxidase (MnP) is the most common lignin-modifying peroxidase produced by almost all wood-colonizing basidiomycetes causing white-rot and various soil-colonizing litter-decomposing fungi. Multiple forms of this glycosylated heme protein with molecular weights normally at 40 to 50 kDa are secreted by ligninolytic fungi into their microenvironment. There, MnP preferentially oxidizes manganese(II) ions (Mn2+), always present in wood and soils, into highly reactive Mn3+, which is stabilized by fungal chelators such as oxalic acid. Chelated Mn3+ in turn acts as low-molecular weight, diffusible redox-mediator that attacks phenolic lignin structures resulting in the formation of instable free radicals that tend to disintegrate spontaneously. MnP is capable of oxidizing and depolymerizing natural and synthetic lignins as well as entire lignocelluloses (milled straw or wood, pulp) in cell-free systems (in vitro). In vitro depolymerization is enhanced in the presence of co-oxidants such as thiols (e.g. glutathione) or unsaturated fatty acids and their derivatives (e.g. Tween 80). The review summarizes and discusses different approaches to prove lignin decomposition in vitro and lists, in addition, other recalcitrant substances oxidizible by MnP.

A review is presented related to the biochemistry of lignocellulose transformation. The biodegradation of wood constituents is currently understood as a multienzymatic process with the mediation of small molecules; therefore, this review will focus on the roles of these small molecular compounds and radicals working in concert with enzymes. Wood rotting basidiomycetous fungi penetrate wood and lead to more easily metabolized, carbohydrate constituents of the complex. Having a versatile machinery of enzymes, the white rot fungi are able to attack directly the "lignin barrier." They also use a multienzyme system including so-called "feed back" type enzymes, allowing for simultaneous transformation of both lignin and cellulose. These enzymes may function separately or cooperatively.

Crude and purified manganese peroxidase from the white-rot fungi Nematoloma frowardii and Phlebia radiata catalyzed the partial depolymerization of a [14C-ring]labelled synthetic lignin into water-soluble fragments (30–50%). The in vitro depolymerization of the 14C-labelled lignin was accompanied by a release of 14CO2 ranging from 4 to 6%. Small quantities of the thiol mediator glutathione stimulated the depolymerization of lignin resulting in a mineralization and solubilization of up to 10 and 64%, respectively. Most of the water-soluble substances formed had molecular masses around 0.7 kDa, although a higher-molecular mass fraction was also detectable (>2 kDa). Photometric assays using 2,2′-azinobis(3-ethylbenzothiazolinesulphonate) as an indicator demonstrated that high levels of Mn(III), which were very probably responsible for the depolymerization and mineralization of the 14C-labelled lignin, were adjusted within the first 24 h of incubation. The manganese peroxidase catalyzed depolymerization process was not necessarily dependent on H2O2; also in the absence of the H2O2-generating system glucose/glucose oxidase, effective solubilization and mineralization of lignin dehydrogenation polymerizate occurred, due to the in part superoxide dismutase sensitive, ‘oxidase-like’ activity of MnP which probably produces radical species and peroxides from malonate.

Within a screening program more than 750 fungal strains were tested for their ability to attack a German hard coal (Westerholt Mine). Six strains were selected, which modified the physico-chemical properties of hard coal pieces placed on the overgrown surface of Petri dishes (loss of the compact coal structure, ‘erosion,’ increase in wettability). One of these strains, Coprinus sclerotigenis C142-1, liberated 2-hydroxybiphenyl, alkylated benzenes and polycyclic aromatic hydrocarbons (PAH) from powdered hard coal. It is presumed that most of these compounds were liberated from micropores inside of the hard coal macromolecule. Investigations using hard coal derived asphaltenes indicate that the liberation of hydroxylated biphenyls by C. sclerotigenis is due to a real cleavage of chemical bonds. The cultivation of the white-rot fungus Panus tigrinus on wood shavings coated with asphaltenes led to a decrease of the average molecular weights of these hard coal-derived hydrogenation products.

The solubilization of low-rank coal by fungi is not accompanied by its depolymerization, which was proven by several screening programs. A total number of 728 fungal strains were yet tested for solubilizing a German low-rank coal (lignite). The coal were used oxidatively pretreated (3% H 2 O 2 ) and natively. During the first screening, the nitrogen content of the media were reduced to select fungi, which solubilize coal under nitrogen-limited conditions. Among 480 micromycetous fungi tested, at least ten strains were able to solubilize native low-rank coal pieces on a nitrogen-reduced agar-medium. They all belong to the conidiospores forming deuteromycetes (molds). Under ‘solubilizing’ conditions, only two of these ten strains showed weak extracellular activities (oxidases, peroxidases) toward 2,2′-azinobis(3-ethylbenzthiazoline-6-sulphonate) [ABTS]. In a second screening, 256 wood and litter decaying basidiomycetes, above all white-rot fungi, were tested for their ability to solubilize low-rank coal under ligninolytic conditions: not any strain was able to form black coal droplets or conspicuous diffusion areas. By using a nitrogen-rich medium, a few basidiomycetes showed also coal solubilizing activities, however, their ligninolytic enzymes were inhibited. The ‘typical’ solubilization of low-rank coal (formation of black liquids from solid coal particles) by molds depends mainly on the nitrogen content of the medium (resulting in higher pH values) as well as on the oxidation grade of coal (e.g. H 2 O 2 -pretreatment). Extracellular oxidases and peroxidases, both ligninolytic and nonligninolytic ones, seem to play a minor, (or not any), role for this process.