Production of manganic chelates by laccase from the lignin-degrading fungus Trametes (Coriolus) versicolor.

Many ligninolytic basidiomycete fungi have been shown to secrete a group of peroxidase isozymes whose sole function appears to be the peroxide-dependent oxidation of manganous [Mn(II)] to manganic [Mn(III)] ions. Manganic chelates and these Mn peroxidases have been implicated as central to the degradation of various natural and synthetic lignins and lignin-containing effluents by white rot (ligninolytic) fungi. Another group of enzymes, the laccases, are commonly secreted by wood-rotting fungi, but are generally regarded as being able to oxidize (and usually polymerize) only phenolic substrates. In this report it is shown that in the presence of appropriate oxidizable phenolic accessory substances or primary substrates, a variety of laccases and peroxidases catalyzing one-electron oxidations can also produce Mn(III) chelates from Mn(II).     

Different fungal manganese-oxidizing peroxidases: a comparison between Bjerkandera sp. and Phanerochaete chrysosporium

Two manganese-oxidizing peroxidases differing in glycosylation degree were purified from fermenter cultures of Bjerkandera sp. They were characterized and compared with the three manganese-oxidizing peroxidase isoenzymes obtained from the well-known ligninolytic fungus Phanerochaete chrysosporium. All the enzymes showed similar molecular masses but those from P. chrysosporium had less acidic isoelectric point. Moreover, the latter strictly required Mn2+ to oxidize phenolic substrates whereas the Bjerkandera peroxidases had both Mn-mediated and Mn-independent activity on phenolic and non-phenolic aromatic substrates. Taking into account these results, and those reported for Bjerkandera adusta and different Pleurotus species, we concluded that two different types of Mn(2+)-oxidizing peroxidases are secreted by ligninolytic fungi.     

Degradation of aromatic compounds through the β‐ketoadipate pathway is required for pathogenicity of the tomato wilt pathogen Fusarium oxysporum f. sp. lycopersici

Plant roots react to pathogen attack by the activation of general and systemic resistance, including the lignification of cell walls and increased release of phenolic compounds in root exudate. Some fungi have the capacity to degrade lignin using ligninolytic extracellular peroxidases and laccases. Aromatic lignin breakdown products are further catabolized via the β‐ketoadipate pathway. In this study, we investigated the role of 3‐carboxy‐cis,cis‐muconate lactonizing enzyme (CMLE), an enzyme of the β‐ketoadipate pathway, in the pathogenicity of Fusarium oxysporum f. sp. lycopersici towards its host, tomato. As expected, the cmle deletion mutant cannot catabolize phenolic compounds known to be degraded via the β‐ketoadipate pathway. In addition, the mutant is impaired in root invasion and is nonpathogenic, even though it shows normal superficial root colonization. We hypothesize that the β‐ketoadipate pathway in plant‐pathogenic, soil‐borne fungi is necessary to degrade phenolic compounds in root exudate and/or inside roots in order to establish disease.     

Fungal laccases - occurrence and properties

Laccases of fungi attract considerable attention due to their possible involvement in the transformation of a wide variety of phenolic compounds including the polymeric lignin and humic substances. So far, more than a 100 enzymes have been purified from fungal cultures and characterized in terms of their biochemical and catalytic properties. Most ligninolytic fungal species produce constitutively at least one laccase isoenzyme and laccases are also dominant among ligninolytic enzymes in the soil environment. The fact that they only require molecular oxygen for catalysis makes them suitable for biotechnological applications for the transformation or immobilization of xenobiotic compounds.     

A search for ligninolytic peroxidases in the fungus pleurotus eryngii involving alpha-keto-gamma-thiomethylbutyric acid and lignin model dimers

Because there is some controversy concerning the ligninolytic enzymes produced by Pleurotus species, ethylene release from alpha-keto-gamma-thiomethylbutyric acid (KTBA), as described previously for Phanerochaete chrysosporium lignin peroxidase (LiP), was used to assess the oxidative power of Pleurotus eryngii cultures and extracellular proteins. Lignin model dimers were used to confirm the ligninolytic capabilities of enzymes isolated from liquid and solid-state fermentation (SSF) cultures. Three proteins that oxidized KTBA in the presence of veratryl alcohol and H2O2 were identified (two proteins were found in liquid cultures, and one protein was found in SSF cultures). These proteins are versatile peroxidases that act on Mn2+, as well as on simple phenols and veratryl alcohol. The two peroxidases obtained from the liquid culture were able to degrade a nonphenolic beta-O-4 dimer, yielding veratraldehyde, as well as a phenolic dimer which is not efficiently oxidized by P. chrysosporium peroxidases. The former reaction is characteristic of LiP. The third KTBA-oxidizing peroxidase oxidized only the phenolic dimer (in the presence of Mn2+). Finally, a fourth Mn2+-oxidizing peroxidase was identified in the SSF cultures on the basis of its ability to oxidize KTBA in the presence of Mn2+. This enzyme is related to the Mn-dependent peroxidase of P. chrysosporium because it did not exhibit activity with veratryl alcohol and Mn-independent activity with dimers. These results show that P. eryngii produces three types of peroxidases that have the ability to oxidize lignin but lacks a typical LiP. Similar enzymes (in terms of N-terminal sequence and catalytic properties) are produced by other Pleurotus species. Some structural aspects of P. eryngii peroxidases related to the catalytic properties are discussed.     

Cyclometalated ruthenium(II) complexes as efficient redox mediators in peroxidase catalysis

Cyclometalated ruthenium(II) complexes, [Ru(II)(C~N)(N~N)(2)]PF(6) [HC~N=2-phenylpyridine (Hphpy) or 2-(4'-tolyl)pyridine; N~N=2,2'-bipyridine, 1,10-phenanthroline, or 4,4'-dimethyl-2,2'-bipyridine], are rapidly oxidized by H(2)O(2) catalyzed by plant peroxidases to the corresponding Ru(III) species. The commercial isoenzyme C of horseradish peroxidase (HRP-C) and two recently purified peroxidases from sweet potato (SPP) and royal palm tree (RPTP) have been used. The most favorable conditions for the oxidation have been evaluated by varying the pH, buffer, and H(2)O(2) concentrations and the apparent second-order rate constants ( k(app)) have been measured. All the complexes studied are oxidized by HRP-C at similar rates and the rate constants k(app) are identical to those known for the best substrates of HRP-C (10(6)-10(7) M(-1) s(-1)). Both cationic (HRP-C) and anionic (SPP and RPTP) peroxidases show similar catalytic efficiency in the oxidation of the Ru(II) complexes. The mediating capacity of the complexes has been evaluated using the SPP-catalyzed co-oxidation of [Ru(II)(phpy)(bpy)(2)]PF(6) and catechol as a poor peroxidase substrate as an example. The rate of enzyme-catalyzed oxidation of catechol increases more than 10000-fold in the presence of the ruthenium complex. A simple routine for calculating the rate constant k(c) for the oxidation of catechol by the Ru(III) complex generated enzymatically from [Ru(II)(phpy)(bpy)(2)](+) is proposed. It is based on the accepted mechanism of peroxidase catalysis and involves spectrophotometric measurements of the limiting Ru(II) concentration at different concentrations of catechol. The calculated k(c) value of 0.75 M(-1) s(-1) shows that the cyclometalated Ru(II) complexes are efficient mediators in peroxidase catalysis.     

Bacterial laccases

Laccases (benzenediol oxygen oxidoreductases, EC 1.10.3.2) are polyphenol oxidases (PPO) that catalyze the oxidation of various substituted phenolic compounds by using molecular oxygen as the electron acceptor. The ability of laccases to act on a wide range of substrates makes them highly useful biocatalysts for various biotechnological applications. To date, laccases have mostly been isolated and characterized from plants and fungi, and only fungal laccases are used currently in biotechnological applications. In contrast, little is known about bacterial laccases, although recent rapid progress in the whole genome analysis suggests that the enzymes are widespread in bacteria. Since bacterial genetic tools and biotechnological processes are well established, so developing bacterial laccases would be significantly important. This review summarizes the distribution of laccases among bacteria, their functions, comparison with fungal laccases and their applications.     

Relaxometric evaluation of novel manganese(II) complexes for application as contrast agents in magnetic resonance imaging

Three novel Mn(II) complexes bearing benzyloxymethyl functionalities are reported and their ability to enhance water (1H and 17O) relaxation times is investigated in detail. Two of them contain one coordinated water molecule and display relaxivity values only slightly smaller than those shown by the most clinically used contrast agents (e.g. [Gd(DTPA)(H2O)]2-). Moreover, in these Mn(II) chelates the exchange rate of the coordinated water is ca. one order of magnitude higher if compared to the exchange rates previously reported for Gd(III) complexes with octadentate ligands. The occurrence of such fast exchange rates of the coordinated water is exploited in the formation of macromolecular adducts with human serum albumin to attain systems displaying relaxivity values in the upper range of those so far reported for analogous Gd(III) systems. These results strongly support the view that Mn(II) complexes, in spite of the lower effective magnetic moment, can be considered as viable alternatives to the currently used Gd(III) complexes as contrast agents for MRI applications.     

Ubiquity of lignin-degrading peroxidases among various wood-degrading fungi.

Phanerochaete chrysosporium is rapidly becoming a model system for the study of lignin biodegradation. Numerous studies on the physiology, biochemistry, chemistry, and genetics of this system have been performed. However, P. chrysosporium is not the only fungus to have a lignin-degrading enzyme system. Many other ligninolytic species of fungi, as well as other distantly related organisms which are known to produce lignin peroxidases, are described in this paper. In this study, we demonstrated the presence of the peroxidative enzymes in nine species not previously investigated. The fungi studied produced significant manganese peroxidase activity when they were grown on an oak sawdust substrate supplemented with wheat bran, millet, and sucrose. Many of the fungi also exhibited laccase and/or glyoxal oxidase activity. Inhibitors present in the medium prevented measurement of lignin peroxidase activity. However, Western blots (immunoblots) revealed that several of the fungi produced lignin peroxidase proteins. We concluded from this work that lignin-degrading peroxidases are present in nearly all ligninolytic fungi, but may be expressed differentially in different species. Substantial variability exists in the levels and types of ligninolytic enzymes produced by different white not fungi.     

Comparison of the Vibration Mode of Metals in HNO3 by a Partial Least-Squares Regression Analysis of Near-Infrared Spectra

The near-infrared (NIR) spectra of such metals as Cu(II), Mn(II), Zn(II) and Fe(III) in HNO₃ in the 700–1860 nm region were subjected to a partial least-squares regression analysis and leave-out cross-validation to develop chemometric models. The models yielded a coefficient of determination in cross validation of 0.9744 [Cu(II)], 0.9631 [Mn(II)], 0.9154 [Zn(II)] and 0.741 [Fe(III)]. The regression coefficients for Cu(II), Mn(II) and Zn(II), but not for Fe(III), showed strong negative peaks at around 1050–1200 nm, a zone where spectral bands have been reported to decrease with increasing pH value. A positive peak at around 710–750 nm, which may have been due to water absorption, was observed in regression coefficients of Cu(II), Mn(II) and Zn(II) but not in Fe(III), while a negative peak was observed in that for Fe(III) at around 710–750 nm. These results indicate that the divalent cations [Cu(II), Mn(II) and Zn(II)] showed different absorption in the NIR region from the trivalent cation [Fe(III)], suggesting that the vibration mode of water, which mirrors the interaction between cations and water, may be influenced by valency.     

Comparison of the vibration mode of metals in HNO3 by a partial least-squares regression analysis of near-infrared spectra

The near-infrared (NIR) spectra of such metals as Cu(II), Mn(II), Zn(II) and Fe(III) in HNO(3) in the 700-1,860 nm region were subjected to a partial least-squares regression analysis and leave-out cross-validation to develop chemometric models. The models yielded a coefficient of determination in cross validation of 0.9744 [Cu(II)], 0.9631 [Mn(II)], 0.9154 [Zn(II)] and 0.741 [Fe(III)]. The regression coefficients for Cu(II), Mn(II) and Zn(II), but not for Fe(III), showed strong negative peaks at around 1,050-1,200 nm, a zone where spectral bands have been reported to decrease with increasing pH value. A positive peak at around 710-750 nm, which may have been due to water absorption, was observed in regression coefficients of Cu(II), Mn(II) and Zn(II) but not in Fe(III), while a negative peak was observed in that for Fe(III) at around 710-750 nm. These results indicate that the divalent cations [Cu(II), Mn(II) and Zn(II)] showed different absorption in the NIR region from the trivalent cation [Fe(III)], suggesting that the vibration mode of water, which mirrors the interaction between cations and water, may be influenced by valency.     

Isolation and selection of novel basidiomycetes for decolorization of recalcitrant dyes

Thirty wood-rotting basidiomycetes, most of them causing white rot in wood, were isolated from fruiting bodies growing on decaying wood from the Sierra de Ayllón (Spain). The fungi were identified on the basis of their morphological characteristics and compared for their ability to decolorize Reactive Black 5 and Reactive Blue 38 (as model of azo and phthalocyanine type dyes, respectively) at 75 and 150 mg/L. Only eighteen fungal strains were able to grow on agar plates in the presence of the dyes and only three species (Calocera cornea, Lopharia spadicea, Polyporus alveolaris) decolorized efficiently both dyes at both concentrations. The ligninolytic activities, involved in decolorization dyes (laccases, lignin peroxidases, Mn-oxidizing peroxidases), were followed in glucose basal medium in the presence of enzyme inducers. The results indicate a high variability of the ligninolytic system within white-rot basidiomycetes. These fungal species and their enzymes can represent new alternatives for the study of new biological systems to degrade aromatic compounds causing environmental problems.     

Characterization of reactions catalyzed by manganese peroxidase from Phanerochaete chrysosporium

Manganese peroxidase (MnP) is one of two extracellular peroxidases believed to be involved in lignin biodegradation by the white-rot basidiomycete Phanerochaete chrysosporium. The enzyme oxidizes Mn(II) to Mn(III), which accumulates in the presence of Mn(III) stabilizing ligands. The Mn(III) complex in turn can oxidize a variety of organic substrates. The stoichiometry of Mn(III) complex formed per hydrogen peroxide consumed approaches 2:1 as enzyme concentration increases at a fixed concentration of peroxide or as peroxide concentration decreases at a fixed enzyme concentration. Reduced stoichiometry below 2:1 is shown to be due to Mn(III) complex decomposition by hydrogen peroxide. Reaction of Mn(III) with peroxide is catalyzed by Cu(II), which explains an apparent inhibition of MnP by Cu(II). The net decomposition of hydrogen peroxide to form molecular oxygen also appears to be the only observable reaction in buffers that do not serve as Mn(III) stabilizing ligands. The nonproductive decomposition of both Mn(III) and peroxide is an important finding with implications for proposed in vitro uses of the enzyme and for its role in lignin degradation. Steady-state kinetics of Mn(III) tartrate and Mn(III) malate formation by the enzyme are also described in this paper, with results largely corroborating earlier findings by others. Based on a comparison of pH effects on the kinetics of enzymatic Mn(III) tartrate and Mn(III) malate formation, it appears that pH effects are not due to ionizations of the Mn(III) complexing ligand.     

Role of fungal peroxidases in biological ligninolysis

The degradation of lignin by filamentous fungi is a major route for the recycling of photosynthetically fixed carbon, and the oxidative mechanisms employed have potential biotechnological applications. The lignin peroxidases (LiPs), manganese peroxidases (MnPs), and closely related enzymes of white rot basidiomycetes are likely contributors to fungal ligninolysis. Many of them cleave lignin model compounds to give products consistent with those found in residual white-rotted lignin, and at least some depolymerize synthetic lignins. However, none has yet been shown to delignify intact lignocellulose in vitro. The likely reason is that the peroxidases need to act in concert with small oxidants that can penetrate lignified tissues. Recent progress in the dissolution and NMR spectroscopy of plant cell walls may allow new inferences about the nature of the oxidants involved. Furthermore, increasing knowledge about the genomes of ligninolytic fungi may help us decide whether any of the peroxidases has an essential role.     

Lignin and Mn peroxidase-catalyzed oxidation of phenolic lignin oligomers

The oxidation of phenolic oligomers by lignin and manganese peroxidases was studied by transient-state kinetic methods. The reactivity of peroxidase intermediates compound I and compound II was studied with the phenol guaiacol along with a beta-O-4 phenolic dimer, trimer, and tetramer. Compound I of both peroxidases is much more reactive than compound II. The rate constants for these substrates with Mn peroxidase compound I range from 1.0 x 10(5) M-1 s-1 for guaiacol to 1.1 x 10(3) M-1 s-1 for the tetramer. Reactivity is much higher with lignin peroxidase compound I with rate constants ranging from 1.2 x 10(6) M-1s-1 for guaiacol to 3.6 x 10(5) M-1 s-1 for the tetramer. Rate constants with compound II are much lower with Mn peroxidase exhibiting very little reactivity. The rate constants dramatically decreased with both peroxidases as the size of the substrate increased. The extent of the decrease was much more dramatic with Mn peroxidase, leading us to conclude that, despite its ability to oxidize phenols, Mn2+ is the only physiologically significant substrate. The rate decrease associated with increasing substrate size was more gradual with lignin peroxidase. These data indicate that whereas Mn peroxidase cannot efficiently directly oxidize the lignin polymer, lignin peroxidase is well suited for direct oxidation of polymeric lignin.     

Free metal ion depletion by "Good's" buffers. I. N-(2-acetamido)iminodiacetic acid 1:1 complexes with calcium(ii). magnesium(II), zinc(II), manganese(II), cobalt(II), nickel(II), and copper(II).

Formation (affinity) constants for 1:1 complexes of N-(2-acetamido)iminodiacetic acid (ADAH₂) with Ca(II), Mg(II), Mn(II), Zn(II), Co(II), Ni(II), and Cu(II) have been determined. Probable structures of the various metal chelates existing in solution are discussed. Values for the deprotonation of the amide group in [Cu(ADA)] and subsequent hydroxo complex formation are also reported. The use of ADA as a buffer is considered in terms of metal buffers complexes which can be formed at physiological pH, i.e., at pH 7.0 there is essentially no free metal ion in 1:1 M²⁺ to ADA solutions.     

Purification and characterization of pyranose oxidase from the white rot fungus Trametes multicolor

We purified an intracellular pyranose oxidase from mycelial extracts of the white rot fungus Trametes multicolor by using ammonium sulfate fractionation, hydrophobic interaction, ion-exchange chromatography, and gel filtration. The native enzyme has a molecular mass of 270 kDa as determined by equilibrium ultracentrifugation and is composed of four identical 68-kDa subunits as determined by matrix-assisted laser desorption ionization mass spectrometry. Each subunit contains one covalently bound flavin adenine dinucleotide as its prosthetic group. The enzyme oxidizes several aldopyranoses specifically at position C-2, and its preferred electron donor substrates are D-glucose, D-xylose, and L-sorbose. During this oxidation reaction electrons are transferred to oxygen, yielding hydrogen peroxide. In addition, the enzyme catalyzes the two-electron reduction of 1,4-benzoquinone, several substituted benzoquinones, and 2,6-dichloroindophenol, as well as the one-electron reduction of the ABTS [2,2'-azinobis(3-ethylbenzthiazolinesulfonic acid)] cation radical. As judged by the catalytic efficiencies (k(cat)/K(m)), some of these quinone electron acceptors are much better substrates for pyranose oxidase than oxygen. The optimum pH of the pyranose oxidase-catalyzed reaction depends strongly on the electron acceptor employed and varies from 4 to 8. It has been proposed that the main metabolic function of pyranose oxidase is as a constituent of the ligninolytic system of white rot fungi that provides peroxidases with H(2)O(2). An additional function could be reduction of quinones, key intermediates that are formed during mineralization of lignin.     

Use of laccase as a novel, versatile reporter system in filamentous fungi

Laccases are copper-containing enzymes which oxidize phenolic substrates and transfer the electrons to oxygen. Many filamentous fungi contain several laccase-encoding genes, but their biological roles are mostly not well understood. The main interest in laccases in biotechnology is their potential to be used to detoxify phenolic substances. We report here on a novel application of laccases as a reporter system in fungi. We purified a laccase enzyme from the ligno-cellulolytic ascomycete Stachybotrys chartarum. It oxidized the artificial substrate 2,2'-azino-di-(3-ethylbenzthiazolinsulfonate) (ABTS). The corresponding gene was isolated and expressed in Aspergillus nidulans, Aspergillus niger, and Trichoderma reesei. Heterologously expressed laccase activity was monitored in colorimetric enzyme assays and on agar plates with ABTS as a substrate. The use of laccase as a reporter was shown in a genetic screen for the isolation of improved T. reesei cellulase production strains. In addition to the laccase from S. charatarum, we tested the application of three laccases from A. nidulans (LccB, LccC, and LccD) as reporters. Whereas LccC oxidized ABTS (Km = 0.3 mM), LccD did not react with ABTS but with DMA/ADBP (3,5-dimethylaniline/4-amino-2,6-dibromophenol). LccB reacted with DMA/ADBP and showed weak activity with ABTS. The different catalytic properties of LccC and LccD allow simultaneous use of these two laccases as reporters in one fungal strain.     

Potential of selected fungal species to degrade wheat straw,the most abundant plant raw material in Europe

Background

Structural component of plant biomass, lignocellulose, is the most abundant renewable resource in nature. Lignin is the most recalcitrant natural aromatic polymer and its degradation presents great challenge. Nowadays, the special attention is given to biological delignification, the process where white-rot fungi take the crucial place owing to strong ligninolytic enzyme system. However, fungal species, even strains, differ in potential to produce high active ligninolytic enzymes and consequently to delignify plant biomass. Therefore, the goals of the study were characterization of Mn-oxidizing peroxidases and laccases of numerous mushrooms as well as determination of their potential to delignify wheat straw, the plant raw material that, according to annual yield, takes the first place in Europe and the second one in the world.

Results

During wheat straw fermentation, Lentinus edodes HAI 858 produced the most active Mn-dependent and Mn-independent peroxidases (1443.2 U L⁻¹ and 1045.5 U L⁻¹, respectively), while Pleurotus eryngii HAI 711 was the best laccase producer (7804.3 U L⁻¹). Visualized bends on zymogram confirmed these activities and demonstrated that laccases were the dominant ligninolytic enzymes in the studied species. Ganoderma lucidum BEOFB 435 showed considerable ability to degrade lignin (58.5%) and especially hemicellulose (74.8%), while the cellulose remained almost intact (0.7%). Remarkable selectivity in lignocellulose degradation was also noted in Pleurotus pulmonarius HAI 573 where degraded amounts of lignin, hemicellulose and cellulose were in ratio of 50.4%:15.3%:3.8%.

Conclusions

According to the presented results, it can be concluded that white-rot fungi, due to ligninolytic enzymes features and degradation potential, could be important participants in various biotechnological processes including biotransformation of lignocellulose residues/wastes in food, feed, paper and biofuels.

     

Characterization of Laccases and Peroxidases from Wood-Rotting Fungi (Family Coprinaceae)

Panaeolus sphinctrinus, Panaeolus papilionaceus, and Coprinus friesii are described as producers of ligninolytic enzymes. P. papilionaceus and P. sphinctrinus both produced a laccase. In addition, P. sphinctrinus produced a manganese peroxidase. C. friesii secreted a laccase and two peroxidases similar to the peroxidase of Coprinus cinereus. The purified laccases and peroxidases were characterized by broad substrate specificities, significant enzyme activities at alkaline pH values, and remarkably high pH optima. The two peroxidases of C. friesii remained active at pH 7.0 and 60°C for up to 60 min of incubation. The peroxidases were inhibited by sodium azide and ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA), whereas the laccases were inhibited by sodium azide and N,N-diethyldithiocarbamic acid. As determined by native polyacrylamide gel electrophoresis and isoelectric focusing, all three fungi produced laccase isoenzymes.