Lignin-modifying enzymes from selected white-rot fungi: production and role from in lignin degradation

Abstract: White-rot fungi produce extracellular lignin-modifying enzymes, the best characterized of which are laccase (EC 1.10.3.2), lignin peroxidases (EC 1.11.1.7) and manganese peroxidases (EC 1.11.1.7). Lignin biodegradation studies have been carried out mostly using the white-rot fungus Phanerochaete chrysosporium which produces multiple isoenzymes of lignin peroxidase and manganese peroxidase but does not produce laccase. Many other white-rot fungi produce laccase in addition to lignin and manganese peroxidases and in varying combinations. Based on the enzyme production patterns of an array of white-rot fungi, three categories of fungi are suggested: (i) lignin-manganese peroxidase group (e.g. P. chrysosporium and Phlebia radiata ), (ii) manganese peroxidase-laccase group (e.g. Dichomitus squalens and Rigidoporus lignosus ), and (iii) lignin peroxidase-laccase group (e.g. Phlebia ochraceofulva and Junghuhnia separabilima ). The most efficient lignin degraders, estimated by ¹⁴CO₂ evolution from ¹⁴C-[Ring]-labelled synthetic lignin (DHP), belong to the first group, whereas many of the most selective lignin-degrading fungi belong to the second, although only moderate to good [¹⁴C]DHP mineralization is obtained using fungi from this group. The lignin peroxidase-laccase fungi only poorly degrade [¹⁴C]DHP.     

Reevaluation of the manganese requirement for the biobleaching of kraft pulp by white rot fungi

Manganese dependent peroxidase (MnP) is the main enzyme implicated in the biobleaching of kraft pulps by white rot fungi. The goal of this study was to evaluate the Mn requirement for biobleaching of eucalyptus oxygen delignified kraft pulp (OKP) by various white rot fungi: Trametes versicolor, Phanerochaete sordida, Phlebia radiata, Stereum hirsutum and Bjerkandera sp. strain BOS55. All of the strains tested produced MnP and provided extensive bleaching of OKP when 33 μM Mn was included in the medium. Bjerkandera sp. strain BOS55 was the only strain that also displayed MnP production and biobleaching activity of EDTA-extracted OKP in the complete absence of Mn. However, MnP and biobleaching activity in the absence of Mn was dependent on the presence of organic acids in the medium. The fact the biobleaching was correlated to MnP activity irrespective of whether Mn was present or absent suggests that there may be roles for MnP in Bjerkandera under Mn-deficient conditions. Although manganese-independent peroxidase (MIP) and lignin peroxidase (LiP) were also detected, the titres were much smaller in comparison with those of MnP, so their relative role in biobleaching can be predicted to have a minor importance in comparison with MnP. Only in the case of Bjerkandera, was the expression of LiP stimulated in the presence of oxalate but final brightness was not substantially affected.     

Screening of white rot fungi for biobleaching of <Emphasis Type="Italic">Acacia</Emphasis> oxygen-delignified kraft pulp

To reduce the levels of chlorine-based chemicals in Acacia kraft pulp, we sought to isolate white rot fungus strains that could be used for biobleaching. For this purpose, we collected 600 fungal sources from Indonesia and subjected them to a three-step screening method. The first step involved culturing the strains on Acacia mangium wood powder, guaiacol and agar (WGA) medium. Of the 600 sources, 258 strains grew on WGA medium and generated a red color. The second step revealed that 31 of the 258 strains could degrade extractive-free A. mangium wood powder. The third step examined the ability of the strains to bleach A. mangium oxygen-delignified kraft pulp (A-OKP) under various pH conditions and showed that five strains could biobleach A-OKP at pH 5, 6, and 8. In contrast, the biobleaching abilities of Trametes versicolor and Phanerochaete chrysosporium, which served as standards, were much lower than those of the five new strains, particularly at pH 8. These five strains may be useful for biobleaching of A-OKP.     

Role of laccase in lignin degradation by white-rot fungi

Abstract Laccase is commonly found in white-rot fungi and catalyses the abstraction of one electron from the phenolic hydroxyl group to polymerize or depolymerize lignin model compounds. Laccase degrades both β-1 and β-O-4 dimers via C _α - C _β cleavage, C _α oxidation and alkyl-aryl cleavage. Also, aromatic ring cleavage may be detected following the action of laccase. Laccase can also oxidize non-phenolic compounds when primary mediators, such as 2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonate), are co-present. Laccase produces Mn(III) chelates which allow wood-decaying enzymes to penetrate wood cell walls. Laccase is considered to be capable of degrading lignin together with lignin peroxidase and manganese peroxidase.     

Effect of culture conditions on manganese peroxidase production and activity by some white rot fungi

The ligninolytic system of white rot fungi is primarily composed of lignin peroxidase, manganese peroxidase (MnP) and laccase. The present work was carried out to determine the best culture conditions for production of MnP and its activity in the relatively little-explored cultures of Dichomitus squalens, Irpex flavus and Polyporus sanguineus, as compared with conditions for Phanerochaete chrysosporium and Coriolus versicolor. Studies on enzyme production under different nutritional conditions revealed veratryl alcohol, guaiacol, Reax 80 and Polyfon H to be excellent MnP inducers. Electronic Publication     

Biological bleaching of kraft pulps by white-rot fungi and their enzymes

Abstract: The use of white-rot fungi, especially Trametes versicolor and isolate IZU-154, to delignify and brighten kraft pulps is reviewed. The fungal treatments are effective but slow; the responsible enzymes are being studied with a view to accelerating the process. Manganese peroxidase, or laccase with a co-substrate, can demethylate and partially solubilize the lignin in pulps, mimicking the early steps of the fungal delignification.     

Simultaneous biodegradation ofp-cresol and phenol by the basidiomycetePhanerochaete chrysosporium

Summary The fungusPhanerochaete chrysoporium BKM-F-1767 was able to degrade high concentrations ofp-cresol (up to 150 mg L^–1) provided that glucose was added as a carbon and energy source and conditions favourable to ligninolytic enzyme activities were used, i.e. a nitrogen-limited medium. The fungus also simultaneously degradedp-cresol (50 mg L^–1) and phenol (50 mg L^–1) in a mixture at similar rates. Kinetics ofp-cresol biodegradation were almost identical whether the compound was tested individually or in a mixture with phenol.     

Novel heme-containing enzyme possibly involved in lignin degradation by the white-rot fungus Junghuhnia separabilima

Abstract The white-rot fungus Junghuhnia separabilima (Pouz.)Ryv, showed high levels of laccase production in cultures supplemented with veratric acid. Laccase, lignin peroxidase and an unknown peroxidase were separated from the extracellular culture fluid using anion-exchange FPLC. Three laccase species, three lignin peroxidases and a novel heme-containing protein were characterized by gel electrophoresis and isoelectric focusing. The new hemoprotein has a molecular mass of 44 kDa, isoelectric point of 3,4 and pH optimum of 5.5 for oxidation of o -dianisidine in the presence of H₂O₂. However it oxidised diaminobenzidine and guaiacol in the absence of H₂O₂. Veratryl alcohol and phenol red were not substratesfor this enzyme with or without addition of H₂O₂ and Mn(II). In addition the enzyme did not produce H₂O₂.     

Optimization of manganese peroxidase production by the white rot fungus Bjerkandera sp. strain BOS55

Manganese dependent peroxidase (MnP) is the most ubiquitous peroxidase produced by white rot fungi. MnP is known to be involved in lignin degradation, biobleaching and in the oxidation of hazardous organopollutants. Bjerkandera sp. strain BOS55 is a nitrogen-unregulated white rot fungus which produces high amounts of MnP in the excess of N-nutrients due to increased biomass yield. Therefore, the strain is a good candidate for use in large scale production of this enzyme. The objective of this study was to optimize the MnP production in N-sufficient cultures by varying different physiological factors such as Mn concentration, culture pH, incubation temperature and the addition of organic acids. The fungus produced the highest level of MnP (up to 900 U 1⁻¹) when the Mn concentration was 0.2 to 1 mM, the pH value was 5.2, and the incubation temperature was 30°C. A noteworthy finding was that MnP was also produced at lower levels in the complete absence of Mn. The addition of organic acids like glycolate, malonate, glucuronate, gluconate, 2-hydroxybutyrate to the culture medium increased the peak titres of MnP up to 1250 U 1⁻¹. FPLC profiles indicated that the organic acids stimulated the production of all MnP isoenzymes present in the extracellular fluid of the fungus.     

Xylanases of marine fungi of potential use for biobleaching of paper pulp

Microbial xylanases that are thermostable, active at alkaline pH and cellulase-free are generally preferred for biobleaching of paper pulp. We screened obligate and facultative marine fungi for xylanase activity with these desirable traits. Several fungal isolates obtained from marine habitats showed alkaline xylanase activity. The crude enzyme from NIOCC isolate 3 (Aspergillus niger), with high xylanase activity, cellulase-free and unique properties containing 580 U l^–1 xylanase, could bring about bleaching of sugarcane bagasse pulp by a 60 min treatment at 55°C, resulting in a decrease of ten kappa numbers and a 30% reduction in consumption of chlorine during bleaching. The culture filtrate showed peaks of xylanase activity at pH 3.5 and pH 8.5. When assayed at pH 3.5, optimum activity was detected at 50°C, with a second peak of activity at 90°C. When assayed at pH 8.5, optimum activity was seen at 80°C. The crude enzyme was thermostable at 55°C for at least 4 h and retained about 60% activity. Gel filtration of the 50–80% ammonium sulphate-precipitated fraction of the crude culture filtrate separated into two peaks of xylanase with specific activities of 393 and 2,457 U (mg protein)^–1. The two peaks showing xylanase activity had molecular masses of 13 and 18 kDa. Zymogram analysis of xylanase of crude culture filtrate as well as the 50–80% ammonium sulphate-precipitated fraction showed two distinct xylanase activity bands on native PAGE. The crude culture filtrate also showed moderate activities of -xylosidase and -l-arabinofuranosidase, which could act synergistically with xylanase in attacking xylan. This is the first report showing the potential application of crude culture filtrate of a marine fungal isolate possessing thermostable, cellulase-free alkaline xylanase activity in biobleaching of paper pulp.     

Suppressive effect of l-phenylalanine on manganese peroxidase in the white-rot fungus Phanerochaete chrysosporium

Abstract The effect of added l-amino acids and NH₄⁺ on manganese peroxidase activity in ligninolytic cultures of Phanerochaete chrysosporium were investigated. Among 11 amino acids (0.2 mM) tested, including phenylalanine, glutamate, glutamine, histidine, alanine, iso-leucine, ornithine, glycine, aspartate, proline, and arginine, phenylalanine was the most effective in suppression of manganese peroxidase synthesis. However, all the amino acids tested except proline completely suppressed the enzyme synthesis at 2 mM concentration.     

Modification of wheat straw lignin by solid state fermentation with white-rot fungi

The potential of crude enzyme extracts, obtained from solid state cultivation of four white-rot fungi (Trametes versicolor, Bjerkandera adusta, Ganoderma applanatum and Phlebia rufa), was exploited to modify wheat straw cell wall. At different fermentation times, manganese-dependent peroxidase (MnP), lignin peroxidase (LiP), laccase, carboxymethylcellulase (CMCase), avicelase, xylanase and feruloyl esterase activities were screened and the content of lignin as well as hydroxycinnamic acids in fermented straw were determined. All fungi secreted feruloyl esterase while LiP was only detected in crude extracts from B. adusta. Since no significant differences (P > 0.05) were observed in remaining lignin content of fermented straw, LiP activity was not a limiting factor of enzymatic lignin removal process. The levels of esterified hydroxycinnamic acids degradation were considerably higher than previous reports with lignocellulosic biomass. The data show that P. rufa, may be considered for more specific studies as higher ferulic and p-coumaric acids degradation was observed for earlier incubation times.     

Fractionation of subcellular membranes of the secretory pathway from the peroxidase-producing white-rot fungus Phanerochaete chrysosporium

Abstract Mycelia from the basidiomycete Phanerochaete chrysosporium , producing lignin and manganese peroxidases, were homogenized and fractionated on a sucrose gradient. The main subcellular fungal membrane fractions were successfully separated. Lipid composition analyses of the isolated membranes as well as associated marker enzymes distribution gave evidence to similarities with membranes originating from plants. Lignin and manganese peroxidases were investigated by immunodetection in subcellular fractions. Our results show that lignin and manganese peroxidases are mainly associated with Golgi apparatus vesicles and, to a lesser extent, with endoplasmic reticulum and light density vesicles, but not with plasma membranes.     

Production of phenol-oxidizing enzymes in the interaction between white-rot fungi

The role of the phenol-oxidizing enzymes, laccase and peroxidase, was examined in the fungus-to-fungus interaction in dual cultures. Among five white-rot fungi, the following predominance in competition was observed: Pleurotus ostreatus > Trametes versicolor ≧ Pycnoporus coccineus > Ganoderma applanatum > Schizophyllum commune. Both phenol-oxidizing enzyme activities were detected markedly at the confrontation region, and under the mycelia growing over other colonies more than in other areas of the dual culture. This property was most notably observed in the P. ostreatus cultures. The fungi that produce superior active phenol-oxidizing enzymes were predominant in the competition between confronting fungi, indicating that phenol-oxidizing enzymes relate to fungus-to-fungus interaction.     

Biodecolourisation of some industrial dyes by white-rot fungi

Eight white-rot fungal strains were screened for biodecolourisation of eight dyes commercially employed in various industries. Decolourisation of Poly R 478 was used as a standard to ascertain the dye-decolourisation potential of various fungi. All the fungi tested significantly decolourised Poly R 478 on solid agar medium. When tested in a nitrogen-limited broth medium, Dichomitus squalens, Irpex flavus, Phlebia spp. and Polyporus sanguineus were better industrial dye decolourisers than Phanerochaete chrysosporium.     

Purification and characterization of peroxidases from the dye-decolorizing fungus Bjerkandera adusta

A peroxidase oxidizing Mn²⁺ (MnP) is described for the first time in Bjerkandera adusta, a fungus efficiently degrading xenobiotic compounds. The MnP appeared as two isoenzymes, which were purified to homogeneity together with two lignin peroxidases (LiP). Their N-terminal sequences were identical, but the MnP isoenzymes showed more basic isoelectric points and differences in amino acid composition and catalytic properties. The B. adusta LiP is similar to LiP from Phanerochaete chrysosporium. However, the interest of the MnP described here is related to its ability to catalyze Mn²⁺-mediated as well as Mn²⁺-independent reactions on aromatic compounds, which may be of use for applications in biotechnology and environmental technology.     

Discovery and characterization of new O-methyltransferase from the genome of the lignin-degrading fungus Phanerochaete chrysosporium for enhanced lignin degradation

Using bioinformatic homology search tools, this study utilized sequence phylogeny, gene organization and conserved motifs to identify members of the family of O-methyltransferases from lignin-degrading fungus Phanerochaete chrysosporium. The heterologous expression and characterization of O-methyltransferases from P. chrysosporium were studied. The expressed protein utilized S-(5′-adenosyl)-l-methionine p-toluenesulfonate salt (SAM) and methylated various free-hydroxyl phenolic compounds at both meta and para site. In the same motif, O-methyltransferases were also identified in other white-rot fungi including Bjerkandera adusta, Ceriporiopsis (Gelatoporia) subvermispora B, and Trametes versicolor. As free-hydroxyl phenolic compounds have been known as inhibitors for lignin peroxidase, the presence of O-methyltransferases in white-rot fungi suggested their biological functions in accelerating lignin degradation in white-rot basidiomycetes by converting those inhibitory groups into non-toxic methylated phenolic ones.     

Changes to water repellence of soil caused by the growth of white-rot fungi: studies using a novel microcosm system

A microcosm system is described which permits assessment of the progressive growth of filamentous fungi through soil. We report on its application to measure the effects of Coriolus versicolor and Phanerochaete chrysosporium upon the sorptivity and water repellence of a mineral soil, measured using a miniature infiltration device. Both fungal species caused moderate sub-critical repellence. Since the pore structure was unaffected, the repellence was probably due to hydrophobic substances of fungal origin. This is the first report of changes in soil repellence caused by the growth of potential xenobiotic bioremediating fungi. The potential consequences are discussed.     

Degradation of anthracene by selected white rot fungi

Abstract Approximately 60% of the originally supplied anthracene (AC) was degraded in ligninolytic stationary cultures of selected white rot fungi within 21 days. All the white rot fungi tested oxidized AC to anthraquinone (AQ). Unlike Phanerochaete chrysosporium and strain Px, with Pleurotus ostreatus, Coriolopsis polyzona and Trametes versicolor , AQ did not accumulate in the cultures, indicating that AQ was degraded further and its degradation did not appear to be a rate-limiting step. However, P. ostreatus and C. polyzona failed to degrade AQ in the absence of AC. P. ostreatus, T. versicolor and strain Px did not produce lignin peroxidase (ligninase) (LIP) under the test conditions but oxidized AC to AQ suggesting that white rot fungi produce enzyme(s) other than LIP capable of oxidizing compounds with high ionization potential like AC. Moreover, in the case of Ph. chrysosporium and C. polyzona , AC degradation started earlier than the production of LIP. Veratryl alcohol (VA) seemed to be playing a role in AC oxidation catalyzed by LIP in Ph. chrysosporium .     

Molecular Evolution and Diversity of Lignin Degrading Heme Peroxidases in the Agaricomycetes

The plant and microbial peroxidase superfamily encompasses three classes of related protein families. Class I includes intracellular peroxidases of prokaryotic origin, class II includes secretory fungal peroxidases, including the lignin degrading enzymes manganese peroxidase (MnP), lignin peroxidase (LiP), and versatile peroxidase (VP), and class III includes the secretory plant peroxidases. Here, we present phylogenetic analyses using maximum parsimony and Bayesian methods that address the origin and diversification of class II peroxidases. Higher-level analyses used published full-length sequences from all members of the plant and microbial peroxidase superfamily, while lower-level analyses used class II sequences only, including 43 new sequences generated from Agaricomycetes (mushroom-forming fungi and relatives). The distribution of confirmed and proposed catalytic sites for manganese and aromatic compounds in class II peroxidases, including residues supposedly involved in three different long range electron transfer pathways, was interpreted in the context of phylogenies from the lower-level analyses. The higher-level analyses suggest that class II sequences constitute a monophyletic gene family within the plant and microbial peroxidase superfamily, and that they have diversified extensively in the basidiomycetes. Peroxidases of unknown function from the ascomycete Magnaporthe grisea were found to be the closest relatives of class II sequences and were selected to root class II sequences in the lower-level analyses. LiPs evidently arose only once in the Polyporales, which harbors many white-rot taxa, whereas MnPs and VPs are more widespread and may have multiple origins. Our study includes the first reports of partial sequences for MnPs in the Hymenochaetales and Corticiales.