Fungal dye-decolorizing peroxidase diversity: roles in either intra- or extracellular processes.

Fungal dye-decolorizing peroxidases (DyPs) have found applications in the treatment of dye-contaminated industrial wastes or to improve biomass digestibility. Their roles in fungal biology are uncertain, although it has been repeatedly suggested that they could participate in lignin degradation and/or modification. Using a comprehensive set of 162 fully sequenced fungal species, we defined seven distinct fungal DyP clades on basis of a sequence similarity network. Sequences from one of these clades clearly diverged from all others, having on average the lower isoelectric points and hydropathy indices, the highest number of N-glycosylation sites, and N-terminal sequence peptides for secretion. Putative proteins from this clade are absent from brown-rot and ectomycorrhizal species that have lost the capability of degrading lignin enzymatically. They are almost exclusively present in white-rot and other saprotrophic Basidiomycota that digest lignin enzymatically, thus lending support for a specific role of DyPs from this clade in biochemical lignin modification. Additional nearly full-length fungal DyP genes were isolated from the environment by sequence capture by hybridization; they all belonged to the clade of the presumably secreted DyPs and to another related clade. We suggest focusing our attention on the presumably intracellular DyPs from the other clades, which have not been characterized thus far and could represent enzyme proteins with novel catalytic properties. KEY POINTS: • A fungal DyP phylogeny delineates seven main sequence clades. • Putative extracellular DyPs form a single clade of Basidiomycota sequences. • Extracellular DyPs are associated to white-rot fungi.

The genome sequence of the commercially cultivated mushroom Agrocybe aegerita reveals a conserved repertoire of fruiting-related genes and a versatile suite of biopolymer-degrading enzymes.

BACKGROUND: Agrocybe aegerita is an agaricomycete fungus with typical mushroom features, which is commercially cultivated for its culinary use. In nature, it is a saprotrophic or facultative pathogenic fungus causing a white-rot of hardwood in forests of warm and mild climate. The ease of cultivation and fructification on solidified media as well as its archetypal mushroom fruit body morphology render A. aegerita a well-suited model for investigating mushroom developmental biology. RESULTS: Here, the genome of the species is reported and analysed with respect to carbohydrate active genes and genes known to play a role during fruit body formation. In terms of fruit body development, our analyses revealed a conserved repertoire of fruiting-related genes, which corresponds well to the archetypal fruit body morphology of this mushroom. For some genes involved in fruit body formation, paralogisation was observed, but not all fruit body maturation-associated genes known from other agaricomycetes seem to be conserved in the genome sequence of A. aegerita. In terms of lytic enzymes, our analyses suggest a versatile arsenal of biopolymer-degrading enzymes that likely account for the flexible life style of this species. Regarding the amount of genes encoding CAZymes relevant for lignin degradation, A. aegerita shows more similarity to white-rot fungi than to litter decomposers, including 18 genes coding for unspecific peroxygenases and three dye-decolourising peroxidase genes expanding its lignocellulolytic machinery. CONCLUSIONS: The genome resource will be useful for developing strategies towards genetic manipulation of A. aegerita, which will subsequently allow functional genetics approaches to elucidate fundamentals of fruiting and vegetative growth including lignocellulolysis.

The wood rot ascomycete Xylaria polymorpha produces a novel GH78 glycoside hydrolase that exhibits α-L-rhamnosidase and feruloyl esterase activities and releases hydroxycinnamic acids from lignocelluloses.

Soft rot (type II) fungi belonging to the family Xylariaceae are known to substantially degrade hardwood by means of their poorly understood lignocellulolytic system, which comprises various hydrolases, including feruloyl esterases and laccase. In the present study, several members of the Xylariaceae were found to exhibit high feruloyl esterase activity during growth on lignocellulosic materials such as wheat straw (up to 1,675 mU g(-1)) or beech wood (up to 80 mU g(-1)). Following the ester-cleaving activity toward methyl ferulate, a hydrolase of Xylaria polymorpha was produced in solid-state culture on wheat straw and purified by different steps of anion-exchange and size-exclusion chromatography to apparent homogeneity (specific activity, 2.2 U mg(-1)). The peptide sequence of the purified protein deduced from the gene sequence and verified by de novo peptide sequencing shows high similarity to putative α-L-rhamnosidase sequences belonging to the glycoside hydrolase family 78 (GH78; classified under EC 3.2.1.40). The purified enzyme (98 kDa by SDS-PAGE, 103 kDa by size-exclusion chromatography; pI 3.7) converted diverse glycosides (e.g., α-L-rhamnopyranoside and α-L-arabinofuranoside) but also natural and synthetic esters (e.g., chlorogenic acid, hydroxycinnamic acid glycoside esters, veratric acid esters, or p-nitrophenyl acetate) and released free hydroxycinnamic acids (ferulic and coumaric acid) from arabinoxylan and milled wheat straw. These catalytic properties strongly suggest that X. polymorpha GH78 is a multifunctional enzyme. It is the first fungal enzyme that combines glycosyl hydrolase with esterase activities and may help this soft rot fungus to degrade lignocelluloses.

Bioconversion of Lignocellulosic Materials with the Contribution of a Multifunctional GH78 Glycoside Hydrolase from Xylaria polymorpha to Release Aromatic Fragments and Carbohydrates.

A bifunctional glycoside hydrolase GH78 from the ascomycete Xylaria polymorpha (XpoGH78) possesses catalytic versatility towards both glycosides and esters, which may be advantageous for the efficient degradation of the plant cell-wall complex that contains both diverse sugar residues and esterified structures. The contribution of XpoGH78 to the conversion of lignocellulosic materials without any chemical pretreatment to release the water-soluble aromatic fragments, carbohydrates, and methanol was studied. The disintegrating effect of enzymatic lignocellulose treatment can be significantly improved by using different kinds of hydrolases and phenoloxidases. The considerable changes in low (3 kDa), medium (30 kDa), and high (> 200 kDa) aromatic fragments were observed after the treatment with XpoGH78 alone or with this potent cocktail. Synergistic conversion of rape straw also resulted in a release of 17.3 mg of total carbohydrates (e.g., arabinose, galactose, glucose, mannose, xylose) per gram of substrate after incubating for 72 h. Moreover, the treatment of rape straw with XpoGH78 led to a marginal methanol release of approximately 17 µg/g and improved to 270 µg/g by cooperation with the above accessory enzymes. In the case of beech wood conversion, the combined catalysis by XpoGH78 and laccase caused an effect comparable with that of fungal strain X. polymorpha in woody cultures concerning the liberation of aromatic lignocellulose fragments.

DyP-like peroxidases of the jelly fungus Auricularia auricula-judae oxidize nonphenolic lignin model compounds and high-redox potential dyes.

The jelly fungus Auricularia auricula-judae produced an enzyme with manganese-independent peroxidase activity during growth on beech wood (approximately 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 (Mn(2+)) were not oxidized. Most remarkable, however, is the finding that both AjPs oxidized nonphenolic lignin model compounds (veratryl alcohol; adlerol, a nonphenolic beta-O-4 lignin model dimer) at low pH (maximum activity at pH 1.4), which indicates a certain ligninolytic activity of dye-decolorizing peroxidases.

Oxidoreductases on their way to industrial biotransformations.

Fungi produce heme-containing peroxidases and peroxygenases, flavin-containing oxidases and dehydrogenases, and different copper-containing oxidoreductases involved in the biodegradation of lignin and other recalcitrant compounds. Heme peroxidases comprise the classical ligninolytic peroxidases and the new dye-decolorizing peroxidases, while heme peroxygenases belong to a still largely unexplored superfamily of heme-thiolate proteins. Nevertheless, basidiomycete unspecific peroxygenases have the highest biotechnological interest due to their ability to catalyze a variety of regio- and stereo-selective monooxygenation reactions with H2O2 as the source of oxygen and final electron acceptor. Flavo-oxidases are involved in both lignin and cellulose decay generating H2O2 that activates peroxidases and generates hydroxyl radical. The group of copper oxidoreductases also includes other H2O2 generating enzymes - copper-radical oxidases - together with classical laccases that are the oxidoreductases with the largest number of reported applications to date. However, the recently described lytic polysaccharide monooxygenases have attracted the highest attention among copper oxidoreductases, since they are capable of oxidatively breaking down crystalline cellulose, the disintegration of which is still a major bottleneck in lignocellulose biorefineries, along with lignin degradation. Interestingly, some flavin-containing dehydrogenases also play a key role in cellulose breakdown by directly/indirectly "fueling" electrons for polysaccharide monooxygenase activation. Many of the above oxidoreductases have been engineered, combining rational and computational design with directed evolution, to attain the selectivity, catalytic efficiency and stability properties required for their industrial utilization. Indeed, using ad hoc software and current computational capabilities, it is now possible to predict substrate access to the active site in biophysical simulations, and electron transfer efficiency in biochemical simulations, reducing in orders of magnitude the time of experimental work in oxidoreductase screening and engineering. What has been set out above is illustrated by a series of remarkable oxyfunctionalization and oxidation reactions developed in the frame of an intersectorial and multidisciplinary European RTD project. The optimized reactions include enzymatic synthesis of 1-naphthol, 25-hydroxyvitamin D3, drug metabolites, furandicarboxylic acid, indigo and other dyes, and conductive polyaniline, terminal oxygenation of alkanes, biomass delignification and lignin oxidation, among others. These successful case stories demonstrate the unexploited potential of oxidoreductases in medium and large-scale biotransformations.

Influence of Pb contamination in boreal forest soil on the growth and ligninolytic activity of litter-decomposing fungi.

Lignin mineralization activity of three basidiomycetous litter-decomposing fungi (LDF) was studied with humus layer samples taken from a boreal forest soil. The total Pb concentration in the samples was 32,000 mg kg(-1) and water soluble Pb 67 mg kg(-1). Synthetic lignin mineralization by Collybia dryophila and Clitocybe (Lepista) nebularis was strongly inhibited, whereas Stropharia coronilla was more tolerant to Pb stress in soil and liquid cultures. Purified laccases maintained their activity and purified MnPs remained partly active up to a concentration of 1450 mg Pb l(-1). High concentrations of Pb inhibited the growth of LDF and affected the activity of ligninolytic enzymes, but the extent of inhibition varied among different LDF species. In consequence, Pb contamination in soil may have a negative impact on recycling of organic carbon.

5-hydroxymethylfurfural conversion by fungal aryl-alcohol oxidase and unspecific peroxygenase.

Oxidative conversion of 5-hydroxymethylfurfural (HMF) is of biotechnological interest for the production of renewable (lignocellulose-based) platform chemicals, such as 2,5-furandicarboxylic acid (FDCA). To the best of our knowledge, the ability of fungal aryl-alcohol oxidase (AAO) to oxidize HMF is reported here for the first time, resulting in almost complete conversion into 2,5-formylfurancarboxylic acid (FFCA) in a few hours. The reaction starts with alcohol oxidation, yielding 2,5-diformylfuran (DFF), which is rapidly converted into FFCA by carbonyl oxidation, most probably without leaving the enzyme active site. This agrees with the similar catalytic efficiencies of the enzyme with respect to oxidization of HMF and DFF, and its very low activity on 2,5-hydroxymethylfurancarboxylic acid (which was not detected by GC-MS). However, AAO was found to be unable to directly oxidize the carbonyl group in FFCA, and only modest amounts of FDCA are formed from HMF (most probably by chemical oxidation of FFCA by the H2 O2 previously generated by AAO). As aldehyde oxidation by AAO proceeds via the corresponding geminal diols (aldehyde hydrates), the various carbonyl oxidation rates may be related to the low degree of hydration of FFCA compared with DFF. The conversion of HMF was completed by introducing a fungal unspecific heme peroxygenase that uses the H2 O2 generated by AAO to transform FFCA into FDCA, albeit more slowly than the previous AAO reactions. By adding this peroxygenase when FFCA production by AAO has been completed, transformation of HMF into FDCA may be achieved in a reaction cascade in which O2 is the only co-substrate required, and water is the only by-product formed.

Influence of different forest system management practices on leaf litter decomposition rates, nutrient dynamics and the activity of ligninolytic enzymes: a case study from central European forests.

Leaf litter decomposition is the key ecological process that determines the sustainability of managed forest ecosystems, however very few studies hitherto have investigated this process with respect to silvicultural management practices. The aims of the present study were to investigate the effects of forest management practices on leaf litter decomposition rates, nutrient dynamics (C, N, Mg, K, Ca, P) and the activity of ligninolytic enzymes. We approached these questions using a 473 day long litterbag experiment. We found that age-class beech and spruce forests (high forest management intensity) had significantly higher decomposition rates and nutrient release (most nutrients) than unmanaged deciduous forest reserves (P<0.05). The site with near-to-nature forest management (low forest management intensity) exhibited no significant differences in litter decomposition rate, C release, lignin decomposition, and C/N, lignin/N and ligninolytic enzyme patterns compared to the unmanaged deciduous forest reserves, but most nutrient dynamics examined in this study were significantly faster under such near-to-nature forest management practices. Analyzing the activities of ligninolytic enzymes provided evidence that different forest system management practices affect litter decomposition by changing microbial enzyme activities, at least over the investigated time frame of 473 days (laccase, P<0.0001; manganese peroxidase (MnP), P = 0.0260). Our results also indicate that lignin decomposition is the rate limiting step in leaf litter decomposition and that MnP is one of the key oxidative enzymes of litter degradation. We demonstrate here that forest system management practices can significantly affect important ecological processes and services such as decomposition and nutrient cycling.

Experimental approach to follow the spatiotemporal wood degradation in fungal microcosms.

To investigate the spatiotemporal growth dynamics in fungal microcosms and to follow the spatial degradation effects of fungal lignocellulose fermentation, a new and flexible experimental setup was developed and tested. White and brown rot fungi were cultivated under solid-state conditions in beech wood-filled silicon tubes for 5 weeks. After inoculation of wood material at one end of the tube, the culture vessels were aerated and moistured by flushing air through alkaline and aqueous solutions. After incubation, the silicon tubes were harvested and segmented to follow different growth and degradation parameters. This new approach holds great potential since it allows the use of different growth substrates, variable aeration or moisturization conditions and is therefore a useful tool for diverse degradation studies, e.g. respiration/mineralization studies involving flow meters or carbon dioxide sensors or for molecular biological approaches.

Patterns of lignin degradation and oxidative enzyme secretion by different wood- and litter-colonizing basidiomycetes and ascomycetes grown on beech-wood.

The degradation of lignocellulose and the secretion of extracellular oxidoreductases were investigated in beech-wood (Fagus sylvatica) microcosms using 11 representative fungi of four different ecophysiological and taxonomic groups causing: (1) classic white rot of wood (e.g. Phlebia radiata), (2) 'nonspecific' wood rot (e.g. Agrocybe aegerita), (3) white rot of leaf litter (Stropharia rugosoannulata) or (4) soft rot of wood (e.g. Xylaria polymorpha). All strong white rotters produced manganese-oxidizing peroxidases as the key enzymes of ligninolysis (75-2200 mU g(-1)), whereas lignin peroxidase activity was not detectable in the wood extracts. Interestingly, activities of two recently discovered peroxidases - aromatic peroxygenase and a manganese-independent peroxidase of the DyP-type - were detected in the culture extracts of A. aegerita (up to 125 mU g(-1)) and Auricularia auricula-judae (up to 400 mU g(-1)), respectively. The activity of classic peroxidases correlated to some extent with the removal of wood components (e.g. Klason lignin) and the release of small water-soluble fragments (0.5-1.0 kDa) characterized by aromatic constituents. In contrast, laccase activity correlated with the formation of high-molecular mass fragments (30-200 kDa). The differences observed in the degradation patterns allow to distinguish the rot types caused by basidiomycetes and ascomycetes and may be suitable for following the effects of oxidative key enzymes (ligninolytic peroxidases vs. laccases, role of novel peroxidases) during wood decay.

Enhancement of bioconversion of high-molecular mass polycyclic aromatic hydrocarbons in contaminated non-sterile soil by litter-decomposing fungi.

With the focus on alternative microbes for soil-bioremediation, 18 species of litter-decomposing basidiomycetous fungi were screened for their ability to grow on different lignocellulosic substrates including straw, flax and pine bark as well as to produce ligninolytic enzymes, namely laccase and manganese peroxidase. Following characteristics have been chosen as criteria for the strain selection: (i) the ability to grow at least on one of the mentioned materials, (ii) production of either of the ligninolytic enzymes and (iii) the ability to invade non-sterile soil. As the result, eight species were selected for a bioremediation experiment with an artificially contaminated soil (total polycyclic aromatic hydrocarbon (PAH) concentration 250 mg/kg soil). Up to 70%, 86% and 84% of benzo(a)anthracene, benzo(a)pyrene, and dibenzo(a,h)anthracene, respectively, were removed in presence of fungi while the indigenous microorganisms converted merely up to 29%, 26% and 43% of these compounds in 30 days. Low molecular-mass PAHs studied were easily degraded by soil microbes and only anthracene degradation was enhanced by the fungi as well. The agaric basidiomycetes Stropharia rugosoannulata and Stropharia coronilla were the most efficient PAH degraders among the litter-decomposing species used.

The white-rot fungus Cerrena unicolor strain 137 produces two laccase isoforms with different physico-chemical and catalytic properties.

Cerrena unicolor secreted two laccase isoforms with different characteristics during the growth in liquid media. In a synthetic low-nutrient nitrogen glucose medium (Kirk medium), high amounts of laccase (4,000 U l(-1)) were produced in response to Cu2+. Highest laccase levels (19,000 U l(-1)) were obtained in a complex tomato juice medium. The isoforms (Lacc I, Lacc II) were purified to homogeneity with an overall yield of 22%. Purification involved ultrafiltration and Mono Q separation. Lacc I and II had M (w) of 64 and 57 kDa and pI of 3.6 and 3.7, respectively. Both isoforms had an absorption maximum at 608 nm but different pH optima and thermal stability. Optimum pH ranged from 2.5 to 5.5 depending on the substrate. The pH optima of Lacc II were always higher than those of Lacc I. Both laccases were stable at pH 7 and 10 but rapidly lost activity at pH 3. Their temperature optimum was around 60 degrees C, and at 5 degrees C they still reached 30% of the maximum activity. Lacc II was the more thermostable isoform that did not lose any activity during 6 months storage at 4 degrees C. Kinetic constants (K (m), k (cat)) were determined for 2,2'-azino-bis(3-ethylthiazoline-6-sulfonate) (ABTS), 2,6-dimethoxyphenol and syringaldazine.

Involvement of lipid peroxidation in the degradation of a non-phenolic lignin model compound by manganese peroxidase of the litter-decomposing fungus Stropharia coronilla.

Culture liquids of the litter-decomposing basidiomycete Stropharia coronilla showed pro-oxidant activity promoting the peroxidation of linoleic acid. This activity depended on the presence of manganese peroxidase (MnP) in the fungal culture. Pro-oxidant activity maxima coincided with maximum MnP activities during the separation of extracellular proteins by anion-exchange chromatography. Purified MnP1 showed substantial pro-oxidant activity in the presence of acetate and Mn2+ ions, even without the addition of hydrogen peroxide. A non-phenolic beta-O-4 lignin model compound [LMC; 1-(3,4-dimethoxyphenyl)-2-(2-methoxyphenoxy)-1,3-dihydroxypropane] was partially oxidized in an in vitro reaction system developing MnP-dependent lipid peroxidation. The chelating organic acids malonate and tartrate noticeably inhibited both the peroxidation of linoleic acid and the conversion of LMC in the system. The major product of the LMC oxidation was 1-(3,4-dimethoxyphenyl)-1-oxo-2-(2-methoxyphenoxy)-3-hydroxypropane; in addition, small amounts of 3,4-dimethoxybenzaldehyde (veratraldehyde) and 3,4-dimethoxybenzoic (veratric) acid were detected. Thus, MnP-initiated lipid peroxidation may be involved in the degradation of recalcitrant non-phenolic lignin substructures by litter-decomposing fungi similar to MnPs of wood-decaying fungi.

Laccase from the medicinal mushroom Agaricus blazei: production, purification and characterization.

The medicinal mushroom Agaricus blazei produced high amounts of laccase (up to 5,000 units l(-1)) in a complex, agitated liquid medium based on tomato juice, while only traces of the enzyme (<100 units l(-1)) were detected in synthetic glucose-based medium. Purification of the enzyme required three chromatographic steps, including anion and cation exchanging. A. blazei laccase was expressed as a single protein with a molecular mass of 66 kDa and an isoelectric point of 4.0. Spectroscopic analysis of the purified enzyme confirmed that it belongs to the "blue copper oxidases". The enzyme's pH optimum for 2,6-dimethoxyphenol (DMP) and syringaldazine was pH 5.5; but for 2,2'-azino-bis(3-ethylthiazoline-6-sulfonate) (ABTS) no distinct pH optimum was observed (highest activity at the lowest pH tested). Purified laccase was stable at 20 degrees C, pH 7.0 and pH 3.0, but rapidly lost its activity at 40 degrees C or pH 10. Sodium chloride strongly inhibited the enzyme activity, although the inhibition was completely reversible. The following kinetic constants were determined (K(m), k(cat)): 63 microM, 21 s(-1) for ABTS, 4 microM, 5 s(-1) for syringaldazine, 1,026 microM, 15 s(-1) for DMP and 4307 microM, 159 s(-1) for guaiacol. The results show that--in addition to the wood-colonizing white-rot fungi--the typical litter-decomposing basidiomycetes can also produce high titers of laccase in suitable liquid media.