White-rot fungi and their enzymes for the treatment of industrial dye effluents

White-rot fungi produce various isoforms of extracellular oxidases including laccase, Mn peroxidase and lignin peroxidase (LiP), which are involved in the degradation of lignin in their natural lignocellulosic substrates. This ligninolytic system of white-rot fungi (WRF) is directly involved in the degradation of various xenobiotic compounds and dyes. This review summarizes the state of the art in the research and prospective use of WRF and their enzymes (lignin-modifying enzymes, LME) for the treatment of industrial effluents, particularly dye containing effluents. The textile industry, by far the most avid user of synthetic dyes, is in need of ecoefficient solutions for its colored effluents. The decolorization and detoxification potential of WRF can be harnessed thanks to emerging knowledge of the physiology of these organisms as well as of the biocatalysis and stability characteristics of their enzymes. This knowledge will need to be transformed into reliable and robust waste treatment processes.     

Differential expression of manganese peroxidase and laccase in white-rot fungi in the presence of manganese or aromatic compounds

White-rot fungi (basidiomycetes) play an important role in the degradation of lignin which is, beside cellulose, the major compound of wood. This process is catalyzed by ligninolytic enzymes, which are able to cleave oxidatively aromatic rings in lignin structure. Manganese peroxidase and laccase of white-rot-fungi are the most important of these among the ligninolytic enzymes. In addition, they are able to degrade xenobiotic aromatic polymers, persisting as environmental pollutants. Manganese and aromatic compounds have often been discussed as being inducers, enhancers or mediators of these ligninolytic enzymes. It is known that supplementing the growth medium with either Mn²⁺, veratryl alcohol or coal-derived humic acids leads to significantly enhanced extracellular ligninolytic activities. Measuring the amount of expressed mRNA of the two enzymes by quantitative RT-PCR provided evidence that the expression of manganese peroxidase was induced in the three tested white-rot fungi, Clitocybula dusenii b11, Nematoloma frowardii b19, and a straw-degrading strain designated i63–2. Laccase, on the other hand, was expressed in all three fungi with a significant basic activity even without inducer added. However, since the level of laccase mRNA was higher in cultures supplemented with any one of the tested inducers, we conclude that both manganese and the aromatic substances also increase the expression of laccase. Received: 4 February 2000 / Received revision: 11 May 2000 / Accepted: 12 May 2000     

Fungal biodegradation and enzymatic modification of lignin

Lignin, the most abundant aromatic biopolymer on Earth, is extremely recalcitrant to degradation. By linking to both hemicellulose and cellulose, it creates a barrier to any solutions or enzymes and prevents the penetration of lignocellulolytic enzymes into the interior lignocellulosic structure. Some basidiomycetes white-rot fungi are able to degrade lignin efficiently using a combination of extracellular ligninolytic enzymes, organic acids, mediators and accessory enzymes. This review describes ligninolytic enzyme families produced by these fungi that are involved in wood decay processes, their molecular structures, biochemical properties and the mechanisms of action which render them attractive candidates in biotechnological applications. These enzymes include phenol oxidase (laccase) and heme peroxidases [lignin peroxidase (LiP), manganese peroxidase (MnP) and versatile peroxidase (VP)]. Accessory enzymes such as H₂O₂-generating oxidases and degradation mechanisms of plant cell-wall components in a non-enzymatic manner by production of free hydroxyl radicals (·OH) are also discussed.     

Biodegradation of lignocellulosics: microbial, chemical, and enzymatic aspects of the fungal attack of lignin.

Wood is the main renewable material on Earth and is largely used as building material and in paper-pulp manufacturing. This review describes the composition of lignocellulosic materials, the different processes by which fungi are able to alter wood, including decay patterns caused by white, brown, and soft-rot fungi, and fungal staining of wood. The chemical, enzymatic, and molecular aspects of the fungal attack of lignin, which represents the key step in wood decay, are also discussed. Modern analytical techniques to investigate fungal degradation and modification of the lignin polymer are reviewed, as are the different oxidative enzymes (oxidoreductases) involved in lignin degradation. These include laccases, high redox potential ligninolytic peroxidases (lignin peroxidase, manganese peroxidase, and versatile peroxidase), and oxidases. Special emphasis is given to the reactions catalyzed, their synergistic action on lignin, and the structural bases for their unique catalytic properties. Broadening our knowledge of lignocellulose biodegradation processes should contribute to better control of wood-decaying fungi, as well as to the development of new biocatalysts of industrial interest based on these organisms and their enzymes.     

Aryl alcohols in the physiology of ligninolytic fungi

Abstract: White-rot fungi have a versatile machinery of enzymes which work in harmony with secondary aryl alcohol metabolites to degrade the recalcitrant, aromatic biopolymer lignin. This review will focus on the important physiological roles of aryl (veratryl, anisyl and chlorinated anisyl) alcohols in the ligninolytic enzyme system. Their functions include stabilization of lignin peroxidase, charge-transfer reactions and as substrate for oxidases generating extracellular H₂0₂. The aryl alcohol/aldehyde couple is well protected against degradation by the fungi's extracellular ligninolytic enzymes and their concentrations in the extracellular fluid are highly regulated by intracellular enzymes.     

Elimination of Endocrine Disrupting Chemicals using White Rot Fungi and their Lignin Modifying Enzymes: A Review

The ability of white rot fungi (WRF) and their lignin modifying enzymes (LMEs), i.e. laccase and lignin‐ and manganese‐dependent peroxidase, to treat endocrine disrupting chemicals (EDCs) is extensively reviewed in this paper. These chemicals cause adverse health effects by mimicking endogenous hormones in receiving organisms. The alkylphenolic EDCs nonylphenol, bisphenol A and triclosan, the phthalic acid esters dibutylphthalate, diethylphthalate and di‐(2‐ethylhexyl)phthalate, the natural estrogens estrone, 17β‐estradiol, estriol and 17α‐ethynylestradiol and the phytoestrogens genistein and β‐sitosterol have been shown to be eliminated by several fungi and LMEs. WRF have manifested a highly efficient removal of EDCs in aqueous media and soil matrices using both LME and non LME‐systems. The ligninolytic system of WRF could also be used for the elimination of several EDCs and the associated hormone‐mimicking activity. The transformation of EDCs by LMEs and WRF is supported by emerging knowledge on the physiology and biochemistry of these organisms and the biocatalytic properties of their enzymes. Due to field reaction conditions, which drastically differ from laboratory conditions, further efforts will have to be directed towards developing robust and reliable biotechnological processes for the treatment of EDC‐contaminated environmental matrices.     

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.     

Biodegradation of pentachlorophenol by white rot fungi under ligninolytic and nonligninolytic conditions

The roles of lignin peroxidase, manganese peroxidase, and laccase were investigated in the biodegradation of pentachlorophenol (PCP) by several white rot fungi. The disappearance of pentachlorophenol from cultures of wild type strains,P. chrysosporium, Trametes sp. andPleurotus sp., was observed. The activities of manganese peroxidase and laccase were detected inTiametes sp. andPleurotus sp. cultures. However, the activities of ligninolytic enzymes were not detected inP. chrysosporium cultures. Therefore, our results showed that PCP was degraded under ligninolytic as well as nonligninolytic conditions. Indicating that lignin peroxidase, manganese peroxidase, and laccase are not essential in the biodegradation of PCP by white rot fungi.     

Degradation of synthetic lignin by the protoplasts of Phanerochaete chrysosporium in the presence of lignin peroxidase or manganese peroxidase

Lignin was mineralized in the experiments in which ¹⁴C-lignin was incubated with lignin peroxidase or manganese peroxidase in a tartrate buffer in the presence of cycloheximide-treated protoplasts obtained from the ligninolytic mycelia of Phanerochaete chrysosporium. The rate of lignin mineralization was dependent on the lignin peroxidase or manganese peroxidase concentration in the medium. In the experiments in which lignin was incubated with lignin peroxidase or manganese peroxidase, lignin was repolymerized irrespective of the presence of protoplasts mineralizing lignin, suggesting that an active degradation of lignin and repolymerization took place. Taking into account that lignin peroxidase and manganese peroxidase were the only extracellular enzymes in the experiments in which lignin was mineralized by the protoplasts, it is postulated that lignin peroxidase and/or manganese peroxidase can degrade lignin into small fragments which can then be further absorbed by the fungal cells and subsequently degraded to CO₂.     

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.     

白腐菌木质素降解酶及其在木质素降解过程中的相互作用

木质素是一类不易降解的生物物质,在自然界中,白腐真菌对木质素的降解能力最强.白腐真菌降解木质素主要依靠分泌的三种酶:木质素过氧化物酶(Lip)、锰过氧化物酶(MnP)和漆酶(Lac).对白腐真菌分泌的三种木质素降解酶在性质、分布等方面进行了比较,系境地介绍三种木质素降解酶的催化作用,并阐述其在木质素降解过程中的相互作用.     

Depolymerization of low-rank coal by extracellular fungal enzyme systems. II. The ligninolytic enzymes of the coal-humic-acid-depolymerizing fungus Nematoloma frowardii b19

The production of ligninolytic enzymes was studied in surface cultures of the South American white-rot fungus Nematoloma frowardii b19 and four other strains of this ecophysiological group (Clitocybula dusenii b11, Auricularia sp. m37a, wood isolates u39 and u45), which are able to depolymerize low-rank-coal-derived humic acids with the formation of fulvic-acid-like compounds. The fungi produced the three crucial enzymes of lignin degradation – lignin peroxidase, manganese peroxidase and laccase. In the case of N. frowardii b19, laccase and the two peroxidases could be stimulated by veratryl alcohol. Manganese (II) ions (Mn²⁺) caused a rapid increase of Mn peroxidase activity accompanied by the complete repression of lignin peroxidase. Under nitrogen-limited conditions the growth as well as the production of ligninolytic enzymes was partly repressed. During the depolymerization process of coal humic acids using solid agar media, gradients of ligninolytic enzyme activities toward 2,2′-azinobis(3-ethylbenzthiazoline-6-sulphonate) and syringaldazine were detectable inside the agar medium. Received: 5 August 1996 / Received revision: 13 November 1996 / Accepted: 15 November 1996     

Understanding lignin-degrading reactions of ligninolytic enzymes: binding affinity and interactional profile

Previous works have demonstrated that ligninolytic enzymes mediated effective degradation of lignin wastes. The degrading ability greatly relied on the interactions of ligninolytic enzymes with lignin. Ligninolytic enzymes mainly contain laccase (Lac), lignin peroxidase (LiP) and manganese peroxidase (MnP). In the present study, the binding modes of lignin to Lac, LiP and MnP were systematically determined, respectively. Robustness of these modes was further verified by molecular dynamics (MD) simulations. Residues GLU460, PRO346 and SER113 in Lac, residues ARG43, ALA180 and ASP183 in LiP and residues ARG42, HIS173 and ARG177 in MnP were most crucial in binding of lignin, respectively. Interactional analyses showed hydrophobic contacts were most abundant, playing an important role in the determination of substrate specificity. This information is an important contribution to the details of enzyme-catalyzed reactions in the process of lignin biodegradation, which can be used as references for designing enzyme mutants with a better lignin-degrading activity.     

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.     

Distribution of ligninolytic enzymes in selected white-rot fungi

Ten white-rot fungi have been screened for the production of ligninase, manganese peroxidase and laccase. Although the fungi degraded lignin efficiently, they significantly differed in the occurrence of individual ligninolytic enzymes. Based on the enzyme pattern produced under N-limited conditions, the fungi can be divided into the following four groups:1. ligninase-manganese peroxidase-laccase group,2. ligninase-manganese peroxidase group,3. manganese peroxidase-laccase group,4. laccase group.     

Evidence That Ceriporiopsis subvermispora Degrades Nonphenolic Lignin Structures by a One-Electron-Oxidation Mechanism

The white-rot fungus Ceriporiopsis subvermispora is able to degrade nonphenolic lignin structures but appears to lack lignin peroxidase (LiP), which is generally thought to be responsible for these reactions. It is well established that LiP-producing fungi such as Phanerochaete chrysosporium degrade nonphenolic lignin via one-electron oxidation of its aromatic moieties, but little is known about ligninolytic mechanisms in apparent nonproducers of LiP such as C. subvermispora. To address this question, C. subvermispora and P. chrysosporium were grown on cellulose blocks and given two high-molecular-weight, polyethylene glycol-linked model compounds that represent the major nonphenolic arylglycerol-(beta)-aryl ether structure of lignin. The model compounds were designed so that their cleavage via one-electron oxidation would leave diagnostic fragments attached to the polyethylene glycol. One model compound was labeled with (sup13)C at C(inf(alpha)) of its propyl side chain and carried ring alkoxyl substituents that favor C(inf(alpha))-C(inf(beta)) cleavage after one-electron oxidation. The other model compound was labeled with (sup13)C at C(inf(beta)) of its propyl side chain and carried ring alkoxyl substituents that favor C(inf(beta))-O-aryl cleavage after one-electron oxidation. To assess fungal degradation of the models, the high-molecular-weight metabolites derived from them were recovered from the cultures and analyzed by (sup13)C nuclear magnetic resonance spectrometry. The results showed that both C. subvermispora and P. chrysosporium degraded the models by routes indicative of one-electron oxidation. Therefore, the ligninolytic mechanisms of these two fungi are similar. C. subvermispora might use a cryptic LiP to catalyze these C(inf(alpha))-C(inf(beta)) and C(inf(beta))-O-aryl cleavage reactions, but the data are also consistent with the involvement of some other one-electron oxidant.     

Production,partial purification and characterization of ligninolytic enzymes from selected basidiomycetes mushroom fungi

In recent years, many research on the quantity of lignocellulosic waste have been developed. The production, partial purification, and characterisation of ligninolytic enzymes from various fungi are described in this work. On the 21st day of incubation in Potato Dextrose (PD) broth, Hypsizygus ulmarius developed the most laccase (14.83 × 10⁻⁶ IU/ml) and manganese peroxidase (24.11 × 10⁻⁶ IU/ml), while Pleurotus florida produced the most lignin peroxidase (19.56 × ⁻⁶ IU/ml). Laccase (Lac), lignin peroxidase (LiP), and manganese peroxidase (MnP), all generated by selected basidiomycetes mushroom fungi, were largely isolated using ammonium sulphate precipitation followed by dialysis. Laccase, lignin peroxidase, and manganese peroxidase purification findings indicated 1.83, 2.13, and 1.77 fold purity enhancements, respectively. Specific activity of purified laccase enzyme preparations ranged from 305.80 to 376.85 IU/mg, purified lignin peroxidase from 258.51 to 336.95 IU/mg, and purified manganese peroxidase from 253.45 to 529.34 IU/mg. H. ulmarius laccase (376.85 IU/mg) with 1.83 fold purification had the highest specific activity of all the ligninolytic enzymes studied, followed by 2.13 fold purification in lignin peroxidase (350.57 IU/mg) and manganese peroxidase (529.34 IU/mg) with 1.77-fold purification. Three notable bands with molecular weights ranging from 43 to 68 kDa and a single prominent band with a molecular weight of 97.4 kDa were identified on a Native PAGE gel from mycelial proteins of selected mushroom fungus. The SDS PAGE profiles of the mycelial proteins from the selected mushroom fungus were similar to the native PAGE. All three partially purified ligninolytic isozymes display three bands in native gel electrophoresis, with only one prominent band in enzyme activity staining. The 43 kDa, 55 kDa, and 68 kDa protein bands correspond to laccase, lignin peroxidase, and manganese peroxidase, respectively.     

Lignin Peroxidase Activity Is Not Important in Biological Bleaching and Delignification of Unbleached Kraft Pulp by Trametes versicolor

The discovery in 1983 of fungal lignin peroxidases able to catalyze the oxidation of nonphenolic aromatic lignin model compounds and release some CO₂ from lignin has been seen as a major advance in understanding how fungi degrade lignin. Recently, the fungus Trametes versicolor was shown to be capable of substantial decolorization and delignification of unbleached industrial kraft pulps over 2 to 5 days. The role, if any, of lignin peroxidase in this biobleaching was therefore examined. Several different assays indicated that T. versicolor can produce and secrete peroxidase proteins, but only under certain culture conditions. However, work employing a new lignin peroxidase inhibitor (metavanadate ions) and a new lignin peroxidase assay using the dye azure B indicated that secreted lignin peroxidases do not play a role in the T. versicolor pulp-bleaching system. Oxidative activity capable of degrading 2-keto-4-methiolbutyric acid (KMB) appeared unique to ligninolytic fungi and always accompanied pulp biobleaching.     

The selective visualization of lignin peroxidase,manganese peroxidase and laccase,produced by white rot fungi on solid media

A visual method for the selective screening of lignin degrading enzymes, produced by white rot fungi (WRF), was investigated by the addition of coloring additives to solid media. Of the additives used in the enzyme production media, guaiacol and RBBR could be used for the detection of lignin peroxidase (LiP), manganese peroxidase (MnP) and laccase. Syringaldazine and Acid Red 264 were able for the detection of both the MnP and laccase, and the LiP and laccase, respectively, and a combination of these two additives was able to detect each of the ligninases produced by the WRF on solid media.     

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.