Search

Apocarotenoids are among the most highly valued fragrance constituents, being also appreciated as synthetic building blocks. This work shows the ability of unspecific peroxygenases (UPOs, EC1.11.2.1) from several fungi, some of them being described recently, to catalyze the oxyfunctionalization of α- and β-ionones and α- and β-damascones. Enzymatic reactions yielded oxygenated products such as hydroxy, oxo, carboxy, and epoxy derivatives that are interesting compounds for the flavor and fragrance and pharmaceutical industries. Although variable regioselectivity was observed depending on the substrate and enzyme, oxygenation was preferentially produced at the allylic position in the ring, being especially evident in the reaction with α-ionone, forming 3-hydroxy-α-ionone and/or 3-oxo-α-ionone. Noteworthy were the reactions with damascones, in the course of which some UPOs oxygenated the terminal position of the side chain, forming oxygenated derivatives (i.e., the corresponding alcohol, aldehyde, and carboxylic acid) at C-10, which were predominant in the Agrocybe aegeritaUPO reactions, and first reported here.

Deadwood provides habitat for fungi and serves diverse ecological functions in forests. We already have profound knowledge of fungal assembly processes, physiological and enzymatic activities, and resulting physico-chemical changes during deadwood decay. However, in situdetection and identification methods, fungal origins, and a mechanistic understanding of the main lignocellulolytic enzymes are lacking. This study used metaproteomics to detect the main extracellular lignocellulolytic enzymes in 12 tree species in a temperate forest that have decomposed for 8 ½ years. Mainly white-rot (and few brown-rot) Basidiomycota were identified as the main wood decomposers, with Armillariaas the dominant genus; additionally, several soft-rot xylariaceous Ascomycota were identified. The key enzymes involved in lignocellulolysis included manganese peroxidase, peroxide-producing alcohol oxidases, laccase, diverse glycoside hydrolases (cellulase, glucosidase, xylanase), esterases, and lytic polysaccharide monooxygenases. The fungal community and enzyme composition differed among the 12 tree species. Ascomycota species were more prevalent in angiosperm logs than in gymnosperm logs. Regarding lignocellulolysis as a function, the extracellular enzyme toolbox acted simultaneously and was interrelated (e.g. peroxidases and peroxide-producing enzymes were strongly correlated), highly functionally redundant, and present in all logs. In summary, our in situstudy provides comprehensive and detailed insight into the enzymatic machinery of wood-inhabiting fungi in temperate tree species. These findings will allow us to relate changes in environmental factors to lignocellulolysis as an ecosystem function in the future.Graphical Abstract

The recently discovered unspecific peroxygenases (UPOs) from the ascomycetes Chaetomium globosumand Humicola insolenswere capable of selectively hydroxylating isophorone to 4-hydroxyisophorone (4HIP) and 4-ketoisophorone (4KIP), which are substrates of interest for the pharmaceutical and flavor-and-fragrance sectors. The model UPO from the basidiomycete Agrocybe aegeritawas less regioselective, forming 7-hydroxyisophorone (and 7-formylisophorone) in addition to 4HIP. However, it was the most stereoselective UPO yielding the S-enantiomer of 4HIP with 88% ee. Moreover, using H. insolensUPO full kinetic resolution of racemic HIP was obtained within only 15 min, with >75% recovery of the R-enantiomer. Surprisingly, the UPOs from two other basidiomycetes, Marasmius rotulaand Coprinopsis cinerea, failed to transform isophorone. The different UPO selectivities were rationalized by computational simulations, in which isophorone and 4HIP were diffused into the enzymes using the adaptive PELE software, and the distances from heme-bound oxygen in H2O2-activated enzyme to different substrate atoms, and the corresponding binding energies were analyzed. Interestingly, for process upscaling, full conversion of 10 mM isophorone was achieved with H. insolensUPO within nine hours, with total turnover numbers up to 5500. These biocatalysts, which only require H2O2for activation, may represent a novel, simple and environmentally-friendly route for the production of isophorone derivatives.

Because of their minimal requirements, substrate promiscuity and product selectivity, fungal peroxygenases are now considered to be the jewel in the crown of C–H oxyfunctionalization biocatalysts. In this work, the crystal structure of the first laboratory-evolved peroxygenase expressed by yeast was determined at a resolution of 1.5 Å. Notable differences were detected between the evolved and native peroxygenase from Agrocybe aegerita, including the presence of a full N-terminus and a broader heme access channel due to the mutations that accumulated through directed evolution. Further mutagenesis and soaking experiments with a palette of peroxygenative and peroxidative substrates suggested dynamic trafficking through the heme channel as the main driving force for the exceptional substrate promiscuity of peroxygenase. Accordingly, this study provides the first structural evidence at an atomic level regarding the mode of substrate binding for this versatile biocatalyst, which is discussed within a biological and chemical context.

This work gives first evidence that the unspecific peroxygenases (UPOs) from the basidiomycetes Agrocybe aegerita(AaeUPO), Coprinopsis cinerea(rCciUPO) and Marasmius rotula(MroUPO) are able to catalyze the regioselective hydroxylation of trans-stilbene to 4,4′-dihydroxy-trans-stilbene (DHS), a resveratrol (RSV) analogue whose preventive effects on cancer invasion and metastasis have very recently been shown. Nearly complete transformation of substrate (yielding DHS) was achieved with the three enzymes tested, using H2O2as the only co-substrate, with AaeUPO showing exceptionally higher total turnover number (200 000) than MroUPO (26 000) and rCciUPO (1400). Kinetic studies demonstrated that AaeUPO was the most efficient enzyme catalyzing stilbene dihydroxylation with catalytic efficiencies (kcat/Km) one and two orders of magnitude higher than those of MroUPO and rCciUPO, so that 4-hydroxystilbene appears to be the best UPO substrate reported to date. In contrast, the peroxygenase from the ascomycete Chaetomium globosum(CglUPO) failed to hydroxylate trans-stilbene at the aromatic ring and instead produced the trans-epoxide in the alkenyl moiety. In addition, stilbenoids such as pinosylvin (Pin) and RSV were tested as substrates for the enzymatic synthesis of RSV from Pin and oxyresveratrol (oxyRSV) from both RSV and Pin. Overall, lower conversion rates and regioselectivities compared with trans-stilbene were accomplished by three of the UPOs, and no conversion was observed with CglUPO. The highest amount of RSV (63% of products) and oxyRSV (78%) were again attained with AaeUPO. True peroxygenase activity was demonstrated by incorporation of 18O from H218O2into the stilbene hydroxylation products. Differences in the number of phenylalanine residues at the heme access channels seems related to differences in aromatic hydroxylation activity, since they would facilitate substrate positioning by aromatic-aromatic interactions. The only ascomycete UPO tested (that of C. globosum) turned out to have the most differing active site (distal side of heme cavity) and reactivity with stilbenes resulting in ethenyl epoxidation instead of aromatic hydroxylation. The above oxyfunctionalizations by fungal UPOs represent a novel and simple alternative to chemical synthesis for the production of DHS, RSV and oxyRSV.

Background: Manganese peroxidases (MnP) and lignin peroxidases (LiP) are haem-including fungal secreted class-II peroxidases, which are interesting oxidoreductases in protein engineering aimed at designing of biocatalysts for lignin and lignocellulose conversion, dye compound degradation, activation of aromatic compounds, and biofuel production. Objective. Recombinant short-type MnP (Pr-MnP3) of the white rot fungus Phlebia radiata, and its manganese- binding site (E40, E44, D186) directed variants were produced and characterized. To allow catalytic applications, enzymatic bleaching of Reactive Blue 5 and conversion of lignin-like compounds by engineered class- II peroxidases were explored. Method: Pr-MnP3 and its variants were expressed in Escherichia coli. The resultant body proteins were lysed, purified and refolded into haem-including enzymes in 6-7% protein recovery, and examined spectroscopically and kinetically. Results: Successful production of active enzymes was attained, with spectral characteristics of high-spin class-II peroxidases. Recombinant Pr-MnP3 demonstrated high affinity to Mn2+, which was noticeably affected by single (D186H/N) and double (E40H+E44H) mutations. Without addition of Mn2+, Pr- MnP3 was able to oxidize ABTS and decolorize Reactive Blue 5. Pc-LiPH8, its Trp-radical site variants, and engineered CiP-LiP demonstrated conversion of veratryl alcohol and dimeric non-phenolic lignin-model compounds (arylglycerol-?-aryl ethers) with production of veratraldehyde, which is evidence for cation radical formation with subsequent Cα-Cβ cleavage. Pc-LiPH8 and CiP variants were able to effectively oxidize and convert the phenolic dimer (guaiacylglycerol-?-guaiacyl ether). Conclusion: Our results demonstrate suitability of engineered MnP and LiP peroxidases for dyedecolorizing, and efficiency of LiP and its variants for activation and degradation of phenolic and nonphenolic lignin-like aryl ether-linked compounds.

Three ascomycetous soft-rot fungi belonging to the family Xylariaceae were found to produce high levels of a p-nitrophenyl acetate-hydrolyzing activity during growth on lignocellulosic materials, i.e., wheat straw and beech wood chips. Accordingly, Kretzschmaria deusta, Morchella elata, Xylaria polymorphawere seemingly most noteworthy acetyl esterase producers and, of which, X. polymorpha(strain A35) was chosen for further studies. Induction study indicated that raw carbohydrate sources, such as beech wood, rape straw, birch wood, and wheat straw, were extremely important for acetyl esterase production. Acetyl esterase of X. polymorphawas produced in solid-state culture on wheat straw and purified by different steps of anion-exchange and size-exclusion chromatography. This purified enzyme (MW= 44 kDa and pIvalues of 3.5–3.6) exhibited the capability to solubilise in vitro beech wood to release water-soluble lignin fragments with molecular masses of 1–3 kDa as analyzed by high performance size exclusion chromatography.

Hydroxylation of vitamin D by Agrocybe aegeritaand Coprinopsis cinereaperoxygenases was investigated in a combined experimental and computational study. 25-Monohydroxylated vitamins D3(cholecalciferol) and D2(ergocalciferol), compounds of high interest in human health and animal feeding, can be obtained through a reaction with both fungal enzymes. Differences in conversion rates, and especially in site selectivity, were observed. To rationalize the results, diffusion of D2and D3on the molecular structure of the two enzymes was performed using the PELE software. In good agreement with experimental conversion yields, simulations indicate more favorable energy profiles for the substrates' entrance in C. cinereathan for A. aegeritaenzyme. On the other hand, GC-MS analyses show that while a full regioselective conversion of D2and D3into the active C25form is catalyzed by C. cinereaperoxygenase, A. aegeritayielded a mixture of the hydroxylated D3products. From the molecular simulations, relative distance distributions between the haem compound I oxygen atom and H24/H25atoms (hydrogens on C24and C25, respectively) were plotted. Results show large populations for O–H25distances below 3 Å for D2and D3in C. cinereain accordance with the high reactivity observed for this enzyme. In A. aegerita, however, cholecalciferol has similar populations (below 3 Å) for O–H25and O–H24, which can justify the hydroxylation observed in C24. In the case of ergocalciferol, due to the bulky methyl group in position C24, very few structures are found with O–H24distances below 3 Å and thus, as expected, the reaction was only observed at the C25position.

Turn a light on: Enantiospecific hydroxylation of nonactivated CH bonds as well as epoxidations of CC bonds are reported using a novel peroxidase from Agrocybe aegerita(AaeAPO). AaeAPO represents a more active and more versatile alternative to the current gold standard, chloroperoxidase. H2O2was produced in situ by photocatalytic reduction of O2using simple flavin adenine mononucleotide (FMN) catalysts.

Reduktion im Licht: In enantiospezifischen Hydroxylierungen nichtaktivierter C‐H‐Bindungen und Epoxidierungen von CC‐Bindungen ist AaeAPO, eine neuartige Peroxidase aus Agrocybe aegerita, eine aktivere und vielseitigere Alternative zu der derzeit führenden Chlorperoxidase. Mit einfachem Flavinadeninmononucleotid (FMN) als Katalysator wurde H2O2in situ durch photokatalytische Reduktion von O2erzeugt.

From the biotechnological viewpoint, the enzymatic disintegration of plant lignocellulosic biomass is a promising goal since it would deliver fermentable sugars for the chemical sector. Cellobiose dehydrogenase (CDH) is a vital component of the extracellular lignocellulose-degrading enzyme system of fungi and has a great potential to improve catalyst efficiency for biomass processing. In the present study, a CDH from a newly isolated strain of the agaricomycete Coprinellus aureogranulatus(CauCDH) was successfully purified with a specific activity of 28.9 U mg−1. This pure enzyme (MW = 109 kDa, pI = 5.4) displayed the high oxidative activity towards β-1–4-linked oligosaccharides. Not least, CauCDH was used for the enzymatic degradation of rice straw without chemical pretreatment. As main metabolites, glucose (up to 165.18 ± 3.19 mg g−1), xylose (64.21 ± 1.22 mg g−1), and gluconic acid (5.17 ± 0.13 mg g−1) could be identified during the synergistic conversion of this raw material with the fungal hydrolases (e.g., esterase, cellulase, and xylanase) and further optimization by using an RSM statistical approach.

Summary: Paecilomyces inflatus isolated from municipal waste compost was found to have cellulolytic activity in several solid and liquid media. This study was done to reveal the multifarious effects of municipal waste compost on endoglucanase activity of P. inflatus. The highest enzyme activities under the conditions of solid-state fermentation were measured in authentic compost samples compared with wood, straw and bran substrates. In surface liquid cultures glucose, cellobiose, xylan, Avicel cellulose, carboxymethylcellulose (CM-cellulose), starch and citrus pectin were used as carbon sources. All carbon sources supported the growth of P. inflatus. However, only CM-cellulose, cellobiose and pectin noticeably stimulated endoglucanase (EG) activity. Further stimulation of EG activity was obtained in cultures containing 1% CM-cellulose as a carbon source by supplementation with low-molecular mass aromatic compounds vanillin, veratric acid and benzoic acid, and with soil humic acid (SHA). SHA and veratric acid were found to be the most efficient elicitors of the cellulolytic activity. P. inflatus was able to utilize nitrate and ammonium as pure nitrogen sources in media containing cellulose.

Linoleic acid, the predominant unsaturated fatty acid (UFA) in the lipids of wood-rotting fungi, was oxidized by manganese peroxidase (MnP) from the white-rot fungusPhlebia radiatathrough a peroxidation mechanism. The peroxidation was markedly stimulated by hydrogen peroxide. UFAs that are substrates for lipid peroxidation and surfactants that emulsify water-insoluble components were essential for the MnP-catalyzed destruction of a nonphenolic β-O-4-linked lignin model compound (LMC). Moreover, both components stimulated the MnP-catalyzed mineralization of14C-labeled synthetic lignin and14C-labeled wheat straw. A high level of destruction was obtained in reaction systems with Tween 80 acting both as surfactant and source of UFAs. The presence of the linoleic acid in reaction systems with MnP and Tween 80 additionally enhanced rate and level of LMC destruction and lignin mineralization. The results indicate that lipid peroxidation may play an important role in lignin biodegradation by wood-rotting basidiomycetes and support the hypothesis of coupling between the processes.

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 concerto with enzymes. Wood rotting basidiomycetous fungi penetrate wood and lead to more easily metabolized, carbohydrate constitutents 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.

SummaryManganese peroxidase preparations (MnP) from the white-rot fungus Nematoloma frowardiiwere able to release 14CO2directly from 14C-labeled milled wheat straw (MWS; total lignin fraction) and milled straw lignin (MSL; dioxane soluble part of MWS). Apart from the formation of 14CO2(4–10 %) the treatment of insoluble MWS and MSL with MnP resulted in the formation of water-soluble 14C-lignin fragments (lignin solubilization, 14–25%). Analyses with gel permeation chromatography (GPC) demonstrated the formation of lignin fragments with predominant molecular masses around 1 kDa. The extent of MWS mineralization and solubilization was enhanced in the presence of reduced glutathione (GSH) acting as thiol mediator, whereas MSL mineralization was not stimulated by GSH. The principle of direct extracellular mineralization of lignin catalyzed by the MnP system may make a significant contribution to the formation of carbon dioxide in lignincellulose containing habitats.

The depolymerization of humic acids (HAs) obtained from low-rank coal (lignite) to fulvic acids (FAs) was investigated in a cell-free system (in vitro) using manganese peroxidase (MnP) from the white-rot fungus Clitocybula dusenii b11. MnP was produced in surface cultures of C. dusenii which were induced with manganese(II) ions (Mn²⁺, 300 μM). The optimum conditions for the action of MnP were determined by varying following parameters of the enzyme assay: i) concentration of Mn²⁺, ii) concentration of hydrogen peroxide (H2O2), iii) pH value and iv) temperature. Optimum parameters determined were used in subsequent in vitro depolymerization studies of humic acids. For that purpose, following parameters of the reaction mixture were additionally varied: concentration of HAs, concentration of the thiol mediator glutathione (GSH), presence and concentration of organic solvents. As the result, following parameters were found to be optimal for the MnP-catalyzed in vitro depolymerization of HAs into low-molecular weight FAs (MnP activity 0.12 U/ml): 250 μg/ml HAs, 1 mM MnCl2, 46 μM/min H2O2 (continuously supplied by glucose oxidase), 600 μM GSH, 4% (v/v) N,N-dimethylformamide (DMF), pH 4.0, and 37 °C.

The in vitro-oxidation of the three- and four-ring polycyclic aromatic hydrocarbons (PAH) anthracene, phenanthrene, pyrene and fluoranthene by preparations of extracellular lignin peroxidase (LiP), manganese peroxidase (MnP) and laccase (Lacc) of the white rot fungus Nematoloma frowardii and by mushroom tyrosinase (Tyr) and horseradish peroxidase (HRP) was investigated. LiP transformed 58.6% of anthracene and 34.2% of pyrene, whereas 31.5% of anthracene and 11.2% pyrene were oxidized by MnP. In the presence of the mediating substances veratryl alcohol (for LiP), GSH (for MnP), and ABTS (for Lacc, Tyr, HRP), the conversion of PAH was enhanced in most cases. Inclusion of PAH-derivatives, known as intermediates or potential dead-end-products of microbial PAH metabolism, in the in vitro-oxidation studies, demonstrated that the hydroxylated PAH metabolites served as substrates for all oxidoreductases tested, whereas PAH-quinones and oxo-metabolites were not transformed.

Preparations of extracellular manganese peroxidase from the white-rot fungus Nematoloma frowardii and the litter decaying fungus Stropharia rugosoannulata converted rapidly the main intermediates of the explosive 2,4,6-trinitrotoluene — the aminonitrotoluenes. In a cell-free system, 2-amino-4,6-dinitrotoluene, 4-amino-2,6-dinitrotoluene and 2,6-diamino-4-nitrotoluene were degraded without formation of identifiable metabolites. Radioactive experiments using a complex mixture of uniform ring-labeled ¹⁴C-TNT reduction products demonstrated the partial direct mineralization of these compounds by manganese peroxidase. The extent of aminonitrotoluene conversion as well as the release of ¹⁴CO2 from TNT reduction products were considerably enhanced in the presence of thiols like reduced glutathione or the amino acid L-cystein, which probably act as secondary mediators.

The depolymerization of humic acids (HAs) obtained from low-rank coal (lignite) to fulvic acids (FAs) was investigated in a cell-free system (in vitro) using manganese peroxidase (MnP) from the white-rot fungus Clitocybula dusenii b11. MnP was produced in surface cultures of C. dusenii which were induced with manganese(II) ions (Mn²⁺, 300 μM). The optimum conditions for the action of MnP were determined by varying following parameters of the enzyme assay: i) concentration of Mn²⁺, ii) concentration of hydrogen peroxide (H2O2), iii) pH value and iv) temperature. Optimum parameters determined were used in subsequent in vitro depolymerization studies of humic acids. For that purpose, following parameters of the reaction mixture were additionally varied: concentration of HAs, concentration of the thiol mediator glutathione (GSH), presence and concentration of organic solvents. As the result, following parameters were found to be optimal for the MnP-catalyzed in vitro depolymerization of HAs into low-molecular weight FAs (MnP activity 0.12 U/ml): 250 μg/ml HAs, 1 mM MnCl2, 46 μM/min H2O2 (continuously supplied by glucose oxidase), 600 μM GSH, 4% (v/v) N,N-dimethylformamide (DMF), pH 4.0, and 37 °C.

The in vitro-oxidation of the three- and four-ring polycyclic aromatic hydrocarbons (PAH) anthracene, phenanthrene, pyrene and fluoranthene by preparations of extracellular lignin peroxidase (LiP), manganese peroxidase (MnP) and laccase (Lacc) of the white rot fungus Nematoloma frowardii and by mushroom tyrosinase (Tyr) and horseradish peroxidase (HRP) was investigated. LiP transformed 58.6% of anthracene and 34.2% of pyrene, whereas 31.5% of anthracene and 11.2% pyrene were oxidized by MnP. In the presence of the mediating substances veratryl alcohol (for LiP), GSH (for MnP), and ABTS (for Lacc, Tyr, HRP), the conversion of PAH was enhanced in most cases. Inclusion of PAH-derivatives, known as intermediates or potential dead-end-products of microbial PAH metabolism, in the in vitro-oxidation studies, demonstrated that the hydroxylated PAH metabolites served as substrates for all oxidoreductases tested, whereas PAH-quinones and oxo-metabolites were not transformed.

Preparations of extracellular manganese peroxidase from the white-rot fungus Nematoloma frowardii and the litter decaying fungus Stropharia rugosoannulata converted rapidly the main intermediates of the explosive 2,4,6-trinitrotoluene — the aminonitrotoluenes. In a cell-free system, 2-amino-4,6-dinitrotoluene, 4-amino-2,6-dinitrotoluene and 2,6-diamino-4-nitrotoluene were degraded without formation of identifiable metabolites. Radioactive experiments using a complex mixture of uniform ring-labeled ¹⁴C-TNT reduction products demonstrated the partial direct mineralization of these compounds by manganese peroxidase. The extent of aminonitrotoluene conversion as well as the release of ¹⁴CO2 from TNT reduction products were considerably enhanced in the presence of thiols like reduced glutathione or the amino acid L-cystein, which probably act as secondary mediators.

The depolymerization of humic acids (HAs) obtained from low-rank coal (lignite) to fulvic acids (FAs) was investigated in a cell-free system (in vitro) using manganese peroxidase (MnP) from the white-rot fungus Clitocybula dusenii b11. MnP was produced in surface cultures of C. dusenii which were induced with manganese(II) ions (Mn²⁺, 300 μM). The optimum conditions for the action of MnP were determined by varying following parameters of the enzyme assay: i) concentration of Mn²⁺, ii) concentration of hydrogen peroxide (H2O2), iii) pH value and iv) temperature. Optimum parameters determined were used in subsequent in vitro depolymerization studies of humic acids. For that purpose, following parameters of the reaction mixture were additionally varied: concentration of HAs, concentration of the thiol mediator glutathione (GSH), presence and concentration of organic solvents. As the result, following parameters were found to be optimal for the MnP-catalyzed in vitro depolymerization of HAs into low-molecular weight FAs (MnP activity 0.12 U/ml): 250 μg/ml HAs, 1 mM MnCl2, 46 μM/min H2O2 (continuously supplied by glucose oxidase), 600 μM GSH, 4% (v/v) N,N-dimethylformamide (DMF), pH 4.0, and 37 °C.

The in vitro-oxidation of the three- and four-ring polycyclic aromatic hydrocarbons (PAH) anthracene, phenanthrene, pyrene and fluoranthene by preparations of extracellular lignin peroxidase (LiP), manganese peroxidase (MnP) and laccase (Lacc) of the white rot fungus Nematoloma frowardii and by mushroom tyrosinase (Tyr) and horseradish peroxidase (HRP) was investigated. LiP transformed 58.6% of anthracene and 34.2% of pyrene, whereas 31.5% of anthracene and 11.2% pyrene were oxidized by MnP. In the presence of the mediating substances veratryl alcohol (for LiP), GSH (for MnP), and ABTS (for Lacc, Tyr, HRP), the conversion of PAH was enhanced in most cases. Inclusion of PAH-derivatives, known as intermediates or potential dead-end-products of microbial PAH metabolism, in the in vitro-oxidation studies, demonstrated that the hydroxylated PAH metabolites served as substrates for all oxidoreductases tested, whereas PAH-quinones and oxo-metabolites were not transformed.

Preparations of extracellular manganese peroxidase from the white-rot fungus Nematoloma frowardii and the litter decaying fungus Stropharia rugosoannulata converted rapidly the main intermediates of the explosive 2,4,6-trinitrotoluene — the aminonitrotoluenes. In a cell-free system, 2-amino-4,6-dinitrotoluene, 4-amino-2,6-dinitrotoluene and 2,6-diamino-4-nitrotoluene were degraded without formation of identifiable metabolites. Radioactive experiments using a complex mixture of uniform ring-labeled ¹⁴C-TNT reduction products demonstrated the partial direct mineralization of these compounds by manganese peroxidase. The extent of aminonitrotoluene conversion as well as the release of ¹⁴CO2 from TNT reduction products were considerably enhanced in the presence of thiols like reduced glutathione or the amino acid L-cystein, which probably act as secondary mediators.

The degradation of polycyclic aromatic hydrocarbons by a manganese peroxidase crude preparation of Nematoloma frowardiiwas demonstrated for a mixture of eight different polycyclic aromatic hydrocarbons, and the five individual polycyclic aromatic hydrocarbons phenanthrene, anthracene, pyrene, fluoranthene, and benzo[a]pyrene. Oxidation of polycyclic aromatic hydrocarbons was enhanced by the addition of glutathione, a mediator substance, able to form reactive thiyl radicals. Glutathione‐mediated manganese peroxidase (1.96 U ml−1) was capable of mineralizing [14C]pyrene (7.3%), [14C]anthracene (4.7%), [14C]benzo[a]pyrene (4.0%), [14C]benz[a]anthracene (2.9%), and [14C]phenanthrene (2.5%) in a period of 168 h. This is the first description of direct enzymatic mineralization of polycyclic aromatic hydrocarbons by manganese peroxidase, and indicates their important role in the oxidation of polycyclic aromatic hydrocarbons by wood‐decaying fungi.

The depolymerization of humic acids (HAs) obtained from low-rank coal (lignite) to fulvic acids (FAs) was investigated in a cell-free system (in vitro) using manganese peroxidase (MnP) from the white-rot fungus Clitocybula dusenii b11. MnP was produced in surface cultures of C. dusenii which were induced with manganese(II) ions (Mn²⁺, 300 μM). The optimum conditions for the action of MnP were determined by varying following parameters of the enzyme assay: i) concentration of Mn²⁺, ii) concentration of hydrogen peroxide (H2O2), iii) pH value and iv) temperature. Optimum parameters determined were used in subsequent in vitro depolymerization studies of humic acids. For that purpose, following parameters of the reaction mixture were additionally varied: concentration of HAs, concentration of the thiol mediator glutathione (GSH), presence and concentration of organic solvents. As the result, following parameters were found to be optimal for the MnP-catalyzed in vitro depolymerization of HAs into low-molecular weight FAs (MnP activity 0.12 U/ml): 250 μg/ml HAs, 1 mM MnCl2, 46 μM/min H2O2 (continuously supplied by glucose oxidase), 600 μM GSH, 4% (v/v) N,N-dimethylformamide (DMF), pH 4.0, and 37 °C.

The in vitro-oxidation of the three- and four-ring polycyclic aromatic hydrocarbons (PAH) anthracene, phenanthrene, pyrene and fluoranthene by preparations of extracellular lignin peroxidase (LiP), manganese peroxidase (MnP) and laccase (Lacc) of the white rot fungus Nematoloma frowardii and by mushroom tyrosinase (Tyr) and horseradish peroxidase (HRP) was investigated. LiP transformed 58.6% of anthracene and 34.2% of pyrene, whereas 31.5% of anthracene and 11.2% pyrene were oxidized by MnP. In the presence of the mediating substances veratryl alcohol (for LiP), GSH (for MnP), and ABTS (for Lacc, Tyr, HRP), the conversion of PAH was enhanced in most cases. Inclusion of PAH-derivatives, known as intermediates or potential dead-end-products of microbial PAH metabolism, in the in vitro-oxidation studies, demonstrated that the hydroxylated PAH metabolites served as substrates for all oxidoreductases tested, whereas PAH-quinones and oxo-metabolites were not transformed.

Preparations of extracellular manganese peroxidase from the white-rot fungus Nematoloma frowardii and the litter decaying fungus Stropharia rugosoannulata converted rapidly the main intermediates of the explosive 2,4,6-trinitrotoluene — the aminonitrotoluenes. In a cell-free system, 2-amino-4,6-dinitrotoluene, 4-amino-2,6-dinitrotoluene and 2,6-diamino-4-nitrotoluene were degraded without formation of identifiable metabolites. Radioactive experiments using a complex mixture of uniform ring-labeled ¹⁴C-TNT reduction products demonstrated the partial direct mineralization of these compounds by manganese peroxidase. The extent of aminonitrotoluene conversion as well as the release of ¹⁴CO2 from TNT reduction products were considerably enhanced in the presence of thiols like reduced glutathione or the amino acid L-cystein, which probably act as secondary mediators.

The depolymerization of humic acids (HAs) obtained from low-rank coal (lignite) to fulvic acids (FAs) was investigated in a cell-free system (in vitro) using manganese peroxidase (MnP) from the white-rot fungus Clitocybula dusenii b11. MnP was produced in surface cultures of C. dusenii which were induced with manganese(II) ions (Mn²⁺, 300 μM). The optimum conditions for the action of MnP were determined by varying following parameters of the enzyme assay: i) concentration of Mn²⁺, ii) concentration of hydrogen peroxide (H2O2), iii) pH value and iv) temperature. Optimum parameters determined were used in subsequent in vitro depolymerization studies of humic acids. For that purpose, following parameters of the reaction mixture were additionally varied: concentration of HAs, concentration of the thiol mediator glutathione (GSH), presence and concentration of organic solvents. As the result, following parameters were found to be optimal for the MnP-catalyzed in vitro depolymerization of HAs into low-molecular weight FAs (MnP activity 0.12 U/ml): 250 μg/ml HAs, 1 mM MnCl2, 46 μM/min H2O2 (continuously supplied by glucose oxidase), 600 μM GSH, 4% (v/v) N,N-dimethylformamide (DMF), pH 4.0, and 37 °C.

The in vitro-oxidation of the three- and four-ring polycyclic aromatic hydrocarbons (PAH) anthracene, phenanthrene, pyrene and fluoranthene by preparations of extracellular lignin peroxidase (LiP), manganese peroxidase (MnP) and laccase (Lacc) of the white rot fungus Nematoloma frowardii and by mushroom tyrosinase (Tyr) and horseradish peroxidase (HRP) was investigated. LiP transformed 58.6% of anthracene and 34.2% of pyrene, whereas 31.5% of anthracene and 11.2% pyrene were oxidized by MnP. In the presence of the mediating substances veratryl alcohol (for LiP), GSH (for MnP), and ABTS (for Lacc, Tyr, HRP), the conversion of PAH was enhanced in most cases. Inclusion of PAH-derivatives, known as intermediates or potential dead-end-products of microbial PAH metabolism, in the in vitro-oxidation studies, demonstrated that the hydroxylated PAH metabolites served as substrates for all oxidoreductases tested, whereas PAH-quinones and oxo-metabolites were not transformed.

Preparations of extracellular manganese peroxidase from the white-rot fungus Nematoloma frowardii and the litter decaying fungus Stropharia rugosoannulata converted rapidly the main intermediates of the explosive 2,4,6-trinitrotoluene — the aminonitrotoluenes. In a cell-free system, 2-amino-4,6-dinitrotoluene, 4-amino-2,6-dinitrotoluene and 2,6-diamino-4-nitrotoluene were degraded without formation of identifiable metabolites. Radioactive experiments using a complex mixture of uniform ring-labeled ¹⁴C-TNT reduction products demonstrated the partial direct mineralization of these compounds by manganese peroxidase. The extent of aminonitrotoluene conversion as well as the release of ¹⁴CO2 from TNT reduction products were considerably enhanced in the presence of thiols like reduced glutathione or the amino acid L-cystein, which probably act as secondary mediators.

ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 200 leading journals. To access a ChemInform Abstract, please click on HTML or PDF.

The degradation of polycyclic aromatic hydrocarbons by a manganese peroxidase crude preparation of Nematoloma frowardii was demonstrated for a mixture of eight different polycyclic aromatic hydrocarbons, and the five individual polycyclic aromatic hydrocarbons phenanthrene, anthracene, pyrene, fluoranthene, and benzo[a]pyrene. Oxidation of polycyclic aromatic hydrocarbons was enhanced by the addition of glutathione, a mediator substance, able to form reactive thiyl radicals. Glutathione-mediated manganese peroxidase (1.96 U ml−1) was capable of mineralizing [14C]pyrene (7.3%), [14C]anthracene (4.7%), [14C]benzo[a]pyrene (4.0%), [14C]benz[a]anthracene (2.9%), and [14C]phenanthrene (2.5%) in a period of 168 h. This is the first description of direct enzymatic mineralization of polycyclic aromatic hydrocarbons by manganese peroxidase, and indicates their important role in the oxidation of polycyclic aromatic hydrocarbons by wood-decaying fungi.