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Abstract: This study revealed a dual effect of copper on D. squalens laccase activity, inducing and suppressing. The addition of copper sulfate led to enhancement of the enzyme activity until it reached a maximum value at peak copper sulfate concentration (PC) followed by suppression and even complete termination of the enzyme activity. Maximum laccase yield is a function of fungi tolerance and media composition. Nutrients had a significant impact on copper-independent and copper-induced laccase activity. Some nutrients also improved tolerance of cells and laccase-producing system to copper load. The effect of specific nutrient depended on the ratio of carbon and nitrogen sources. This research can be used to predict the effective copper concentration and nutrient composition to stimulate the laccase production without compromising the fungi growth. The methylated substrates (3-O-methylglucose, methylcellulose) in combination with casein and copper demonstrated high potential to support fungi tolerance and enhanced laccase activity. [Copyright &y& Elsevier]

Abstract: The use of new printing technologies based on flexographic inks hampers ink elimination during paper recycling, making necessary the development of alternative methods. Decolorization of four flexographic inks has been evaluated by using fungal laccases, three of them from basidiomycetes (Trametes villosa, Coriolopsis rigida, and Pycnoporus coccineus), and one from the ascomycete Myceliophthora thermophila in the presence of synthetic and natural mediators. The results obtained showed a higher capacity of the three basidiomycete laccases to decolorize flexographic inks as compared with M. thermophila laccase, a low redox potential laccase. Basidiomycete laccases decolorized inks without mediators at long reaction times, although the presence of natural or synthetic mediators (above all HBT) accelerated the process. On the other hand, M. thermophila laccase was unable to decolorize the inks assayed. The addition of syringyl-type mediators led to medium levels of decolorization except for R48 ink, which was almost completely decolorized. Most decolorization was obtained during the first hours of treatment, when all the basidiomycete laccases were fully active in the presence of mediators. As opposed to other basidiomycete laccases, which become inactive in the presence of HBT after 24h, the enzyme of P. coccineus was not deactivated by this mediator even after 48h. A complete loss of M. thermophila laccase activity was observed at short times with acetosyringone and methyl syringate, the only two mediators able to promote ink decolorization with this enzyme. [Copyright &y& Elsevier]

Qualitative screening of 295 fungi for laccases yielded 125 laccase positive ones, mostly basidiomycetes. Fifty of these were tested for laccase activity at pH 3.0, 4.5 and 6.0. Most showed maximum activity at pH 4.5, a few showed a broad activity range, two were optimal at pH 3.0 and only the mitosporic fungus Beltraniella sp. was best at pH 6. Most of the 25 fungi assayed at three different temperatures had an optimum at 45°C. The basidiomycete Auricularia sp. acted best at 30°C, while three others showed best activity at 60°C. This study shows the potential of screening diverse fungi for laccase with varying pH and temperature preferences for different applications. [ABSTRACT FROM AUTHOR]

Laccases are oxidases that contain several copper atoms, and catalyse single-electron oxidations of phenolic compounds with concomitant reduction of oxygen to water. The enzymes are particularly widespread in ligninolytic basidiomycetes, but also occur in certain prokaryotes, insects and plants. Depending on the species, laccases are involved in various biosynthetic processes contributing to carbon recycling in land ecosystems and the morphogenesis of biomatrices, wherein low-molecular-weight naturally occurring phenols serve as key enzyme substrates. Studies of these in vivo synthetic pathways have afforded new insights into fungal laccase applicability in green synthetic chemistry. Thus, we here review fungal laccase-catalysed oxidations of naturally occurring phenols that are particularly relevant to the synthesis of fine organic chemicals, and we discuss how the discovered synthetic strategies mimic laccase-involved in vivo pathways, thus enhancing the green nature of such reactions. Laccase-catalysed in vivo processes yield several types of biopolymers, including those of cuticles, lignin, polyflavonoids, humus and the melanin pigments, using natural mono- or poly-phenols as building blocks. The in vivo synthetic pathways involve either phenoxyl radical-mediated coupling or cross-linking reactions, and can be adapted to the design of in vitro oxidative processes involving fungal laccases in organic synthesis; the laccase substrates and the synthetic mechanisms reflect in vivo processes. Notably, such in vitro synthetic pathways can also reproduce physicochemical properties (e.g. those of chromophores, and radical-scavenging, hydration and antimicrobial activities) found in natural biomaterials. Careful study of laccase-associated in vivo metabolic pathways has been rewarded by the discovery of novel green applications for fungal laccases. This review comprehensively summarizes the available data on laccase-catalysed biosynthetic pathways and associated applications in fine chemical syntheses. [ABSTRACT FROM AUTHOR]

White-rot basidiomycetes, the most common wood-rotting organisms, are characterized by their ability to produce extracellular oxidative enzymes, among which laccases are regarded as promising catalysts for many biotechnological applications. A significant obstacle to the exploitation of laccase-based bioprocesses is the large amounts of enzyme required. In this study the issue has been addressed by applying a classical breeding approach to increase laccase production yields in the white-rot fungus Pleurotus ostreatus. Starting from two different P. ostreatus varieties, three higher laccase-producing hybrids have been obtained by crossing selected compatible monokaryons. The three selected strains increased the titre of parental strains up to four fold, reaching an expression level of up to 100 000 U/L. One hybrid exhibited a more complex isoenzyme pattern, illustrating the potential of classical breeding to differentiate protein expression. [ABSTRACT FROM AUTHOR]

Fungal laccases have been widely used in industry. The expression of laccase often is repressible by the primary carbon source glucose in many fungi. The underlying basis is largely unclear. We demonstrate here that a gene, TSP2-1, was required for laccase repression by glucose in the basidiomycete Cryptococcus neoformans. TSP2-1 encodes a Tsp2-type tetraspanin. The disruption of TSP2-1 resulted in constant melanin formation and the expression of the laccase gene LAC1. This derepression phenotype was restorable by 10 mM exogenous cyclic AMP (cAMP). A capsule defect in the mutant tsp2-1Δ also was restored by cAMP. The results indicate an interaction of Tsp2-1 with the cAMP-dependent protein kinase A (PKA) pathway that has been shown to modulate laccase repression and capsule biosynthesis in this fungus. Other roles of TSP2-1, e.g., in maintaining cell membrane integrity and stress resistance, also were defined. This work reveals a Tsp2-1-dependent glucose repression in C. neoformans. The function of Tsp2-type tetraspanin Tsp2-1 is described for the first time. [ABSTRACT FROM AUTHOR]

Genetic analysis of basidiomycete Trametes versicolor 1666 from the Komarov Botanical Institute (LEBIN) Collection has been carried out that verified the phylogenetic position of the strain and revealed the presence of a single laccase gene. A study of cultivation dynamics of basidiomycete showed a low level of laccase, peroxidase, and Mn-peroxidase production on glucose-peptone and mineral media. The level of laccase activity in the culture liquid (CL) remained practically unchanged during cultivation in a medium optimized using full factorial experiment as compared to the standard medium. An addition of laccase inducers (syringaldazine, caffeic acid, and synaptic acid) had no effect on the enzyme activity in the culture liquid. Real time PCR showed a lack of reliable difference in the level of laccase gene expression when cultivating the strain under optimal conditions with and without copper ions in the medium. The transcription behavior of laccase gene and occurrence of enzyme activity when cultivating T. versicolor 1666 strain testify to the enzyme constitutive synthesis and the existence of additional regulatory ways in the laccase gene expression. [ABSTRACT FROM AUTHOR]

Genomic DNA encoding the Pleurotus ostreatus LccK laccase was fused with the Coprinopsis cinerea β-tubulin promoter and terminator, and introduced into a C. cinerea strain. Linkage analysis, native PAGE separations, substrate specificity investigations and expression profiling indicated that C. cinerea transformants secrete P. ostreatus LccK, suggesting that the introns of the lccK gene are correctly spliced and the signal peptide for secretion is functional in C. cinerea. Transformants constitutively expressing laccase may be useful for the degradation of aromatic compounds. [ABSTRACT FROM AUTHOR]

Abstract: Tetrabromobisphenol A (TBBPA) degradation was investigated using white rot fungi and their oxidative enzymes. Strains of the Trametes, Pleurotus, Bjerkandera and Dichomitus genera eliminated almost 1mM TBBPA within 4days. Laccase, whose role in TBBPA degradation was demonstrated in fungal cultures, was applied to TBBPA degradation alone and in combination with cellobiose dehydrogenase from Sclerotium rolfsii. Purified laccase from Trametes versicolor degraded approximately 2mM TBBPA within 5h, while the addition of cellobiose dehydrogenase increased the degradation rate to almost 2.5mM within 3h. Laccase was used to prepare TBBPA metabolites 2,6-dibromo-4-(2-hydroxypropane-2-yl) phenol (1), 2,6-dibromo-4-(2-methoxypropane-2-yl) phenol (2) and 1-(3,5-dibromo-4-hydroxyphen-1-yl)-2,2′,6,6′-tetrabromo-4,4′-isopropylidene diphenol (3). As compounds 1 and 3 were identical to the TBBPA metabolites prepared by using rat and human liver fractions (), laccase can provide a simple means of preparing these metabolites for toxicity studies. Products 1 and 2 exhibited estrogenic effects, unlike TBBPA, but lower cell toxicity. [Copyright &y& Elsevier]

Abstract: Different types of feedstocks, including corn stover, wheat straw, soybean straw, switchgrass, and hardwood, were tested to evaluate the effectiveness of fungal pretreatment by Ceriporiopsis subvermispora. After 18-d pretreatment, corn stover, switchgrass, and hardwood were effectively delignified by the fungus through manganese peroxidase and laccase. Correspondingly, glucose yields during enzymatic hydrolysis reached 56.50%, 37.15%, and 24.21%, respectively, which were a 2 to 3-fold increase over those of the raw materials. A further 10–30% increase in glucose yields was observed when pretreatment time extended to 35d. In contrast, cellulose digestibility of wheat straw and soybean straw was not significantly improved by fungal pretreatment. When external carbon sources and enzyme inducers were added during fungal pretreatment of wheat straw and soybean straw, only glucose and malt extract addition improved cellulose digestibility of wheat straw. The cellulose digestibility of soybean straw was not improved. [Copyright &y& Elsevier]

Abstract: Trametes trogii BAFC 463 culture fluids (containing 110Uml−1 laccase; 0.94Uml−1 manganese peroxidase), as well as its purified laccase were capable of decolorizing azoic, indigoid, triphenylmethane, anthraquinonic and heterocyclic dyes, in the absence of redox mediators. Six dyes: RBBR, Indigo Carmine, Xylidine, Malachite Green, Gentian Violet and Bromophenol Blue were almost completely degraded (more than 85% decolorization after 1 d) by either laccase or T. trogii itself in culture, proving the role of the enzyme in dye decolorization. The purified laccase also decolorized 65% of Fast Blue RR and 30% of Azure B and Methylene Blue after 24h. The use of redox mediators significantly increased the decolorization rates (90% decolorization of Azure B after 1h). 1-hydroxybenzotriazole resulted the best redox mediator, but the natural mediator p-hydroxybenzoic acid also demonstrated its efficiency for dye decolorization. Due to their ability to decolorize recalcitrant dyes without addition of redox mediators, high laccase activities, high thermostability and efficient decolorization at 70°C and pH 7.0, even in the presence of high concentrations of heavy metals (100mM Cu+2, Pb+2 or Cd+2) or in a synthetic dyebath, T. trogii culture fluids could be effectively used to decolorize synthetic dyes from effluents. [Copyright &y& Elsevier]

Abstract: The biodiversity–functional relationship in fungal ecology was recently developed and debated, but has rarely been addressed in endophytes. In this study, an integrative culture system was designed to capture a rich fungal consortium from the conifer Abies beshanzuensis. Results indicate an impressive diversity of fungal lineages (a total of 84 taxa classified in Dikarya) and a relatively high proportion of hitherto unknown species (27.4%). The laccase gene was used as a functional marker due to its involvement in lignocellulose degradation. Remarkable diversity of laccase genes was found across a wide range of taxa, with at least 35 and 19 distinct sequences in ascomycetes and basidiomycetes respectively, were revealed. Many groups displayed variable ability to decompose needles. Furthermore, many ascomycetes, including three volatile-producing Muscodor species (Xylariaceae), showed the ability to inhibit pathogens. Notably, most laccase-producing species showed little or no antibiosis and vice versa. Clavicipitalean and ustilaginomycetous fungi, specifically toxic to insects, were inferred from taxonomic information. Intra-specific physiological variation in Pezicula sporulosa, a second dominant species, was clearly high. We conclude that a suite of defensive characteristics in endophytes contributes to improving host fitness under various stresses and that a diversity of laccase genes confers an ecological advantage in competition for nutrients. Intra-specific diversity may be of great ecological significance for ecotypic adaptation. These findings suggest a fair degree of functional complementarity rather than redundancy among endemic symbionts of natural plant populations. [Copyright &y& Elsevier]

Abstract: In a solid state medium using yellow passion fruit waste as substrate, the basidiomycete Ganoderma lucidum produced a laccase as the main ligninolytic enzyme. This crude enzyme presented Michaelian behavior with both substrates tested, namely 3-ethylbenzthiazoline-6-sulphonic acid (ABTS) and the anthraquinone dye remazol brilliant blue R (RBBR). The K M’s for these substrates were, respectively, 0.232×10−3 and 0.602×10−3 M. The actions of NaCl and Na2SO4, two important salts usually found in textile wastewaters, were investigated. The enzyme was inhibited by NaCl, but not by Na2SO4. Inhibition by NaCl was of the mixed type with two different inhibition constants. The enzyme was able to completely decolorize RBBR in the presence of 1.0M Na2SO4 and 50% decolorization was found in the presence of 0.1M NaCl. Such properties certainly make the enzyme a good agent for textile dye effluent treatment considering the fact that wastewaters of this industry usually contain high concentrations of NaCl and Na2SO4. [Copyright &y& Elsevier]

Abstract: Studies of laccases from marine-derived fungi are limited. In the present work, putative laccase genes from three marine-derived basidiomycetes and their laccase activities were evaluated. High amounts of laccase were produced by the fungal strains Marasmiellus sp. CBMAI 1062 (971.2UL−1) and Peniophora sp. CBMAI 1063 (709.03UL−1) when grown for 21d at 28°C in MA2ASW medium prepared with artificial seawater. Marine-derived basidiomycetes produced multiple distinct laccase sequences of about 200bp with 73–90% similarity to terrestrial basidiomycete laccases. Marasmiellus sp. CBMAI 1062 and Tinctoporellus sp. CBMAI 1061 showed the greatest laccase gene diversity with three and four distinct putative laccase sequences, respectively. This is the first report of laccase genes from marine-derived fungi, and our results revealed new putative laccases produced by three basidiomycetes. [ABSTRACT FROM AUTHOR]

Three white-rot fungi displayed a wide diversity in their response to supplemented aromatic compounds. Pyrogallol stimulated Cerrena unicolor laccase and manganese peroxidase (MnP) synthesis in synthetic medium 2.5- and 2-fold, respectively, whereas 2,4,6-trinitrotoluene (TNT) brought about a 2.8-fold increase in laccase yield by Trametes versicolor in submerged fermentation of ethanol production residue. No effect of the tested aromatic compounds on enzyme secretion by Ganoderma lucidum in mannitol-containing medium was detected. Nevertheless, G. lucidum is a potent producer of laccase in submerged fermentation of wheat bran and enzyme synthesis can be further increased by supplementation of medium with an appropriate inducer. The structure and the concentration of aromatic compounds play an important role in the regulation of enzyme synthesis. The supplementation of synthetic medium with 0.03–0.3 mM TNT or hydroquinone increased the differential rate of laccase synthesis by C. unicolor from 1,267 to 3,125–8,630 U mg biomass day. Moreover, the same aromatic compound may function as either an inducer or a repressor, depending on the fungus and enzyme studied. Thus, hydroquinone increased 3-fold T. versicolor laccase activity decreasing 2- and 8-fold the yields of MnP and endoglucanase, respectively. [ABSTRACT FROM AUTHOR]

Three extracellular ligninolytic oxidoreductases that are produced by a commercial laccase-producing Trametes sp. Ha1 were purified and characterized. This fungus showed strong ligninolytic oxidoreductase activity with and without hydrogen peroxide present in the reaction mixture. The oxidoreductase activity was found to be derived from two laccases and a peroxidase. One of the two laccases represents a main component of the commercial laccase preparation from Trametes sp. Ha1. This enzyme had a high thermostability, which makes it attractive for practical applications. The second laccase was induced by the addition of p-xylidine into the culture medium and showed unique characteristics with respect to pI value and substrate specificity. The peroxidase showed wide oxidation activity against aromatic compounds. [Copyright &y& Elsevier]

Two laccase isoenzymes were purified and characterized from the basidiomycete Coriolopsis rigida during transformation of the water-soluble fraction of “alpeorujo” (WSFA), a solid residue derived from the olive oil production containing high levels of toxic compounds. Zymogram assays of laccases secreted by the fungus growing on WSFA and WSFA supplemented with glucose showed two bands with isoelectric points of 3.3 and 3.4. The kinetic studies of the two purified isoenzymes showed similar affinity on 2,6-dimethoxyphenol and 2,2′-azinobis-(3-ethylbenzthiazoline-6-sulfonic acid), used as phenolic and non-phenolic model substrate, respectively. The molecular mass of both proteins was 66 kDa with 9% N-linked carbohydrate. Physico-chemical properties of the purified laccases from media containing WSFA were similar to those obtained from medium with glucose as the main carbon source. In-vitro studies performed with the purified laccases revealed a 42% phenol reduction of WSFA, as well as changes in the molecular mass distribution. These findings indicate that these laccases are involved in the process of transformation, via polymerization by the oxidation of phenolic compounds present in WSFA. A single laccase gene, containing an open reading frame of 1,488 bp, was obtained in PCR amplifications performed with cDNA extracted from mycelia grown on WSFA. The product of the gene shares 90% identity (95% similarity) with a laccase from Trametes trogii and 89% identity (95% similarity) with a laccase from Coriolopsis gallica. This is the first report on purification and molecular characterization of laccases directly involved in the transformation of olive oil residues. [ABSTRACT FROM AUTHOR]

Abstract: Litter decomposing basidiomycetous fungi produce ligninolytic oxidases and peroxidases which are involved in the transformation of lignin, as well as humic and fulvic acids. The aim of this work was to evaluate their importance in lignin transformation in forest litter. Two litter decomposing basidiomycete species differing in their abilities to degrade lignin –Hypholoma fasciculare, and Gymnopus erythropus – were cultured on sterile or non-sterile oak litter and their transformation of a 14C-labelled synthetic lignin (dehydrogenation polymer 14C-DHP) was compared with that of the indigenous litter microflora. Both in sterile and non-sterile litter, colonisation by basidiomycetes led to higher titres of lignocellulose-degrading enzymes, in particular of laccase and Mn-peroxidase (MnP). The titres of the latter were 6 to 40-fold increased in the presence of basidiomycetes compared to non-sterile litter. During 10 weeks, G. erythropus mineralised over 31% of 14C-DHP in sterile litter and 23% in non-sterile litter compared to 14% in the non-sterile control. Lignin mineralization by H. fasciculare was comparable to the non-sterile control, 12% in sterile litter and 16% in the non-sterile litter. The largest part of 14C from 14C-DHP was transformed into humic compounds during litter treatment with both fungi as well as in the control. In addition to the fast lignin mineralization, microcosms containing G. erythropus also showed a lower final content of unaltered lignin and 23–28% of the lignin was converted into water-soluble compounds with relatively low molecular mass (<5kDa). Both G. erythropus and H. fasciculare were also able to further mineralise humic compounds. During a 10-week fungal treatment of an artificial 14C-humic acid (14C-HA) supplemented to the natural humic material of a forest soil, the fungi mineralised 42% and 19% of the labelled material, respectively, under sterile conditions. The 14C-HA mineralization by introduced basidiomycetes in microcosms containing non-sterile humic material, however, did not significantly differ from that of a non-sterile control and was around 12%. Altogether the results show that saprobic basidiomycetes can considerably differ in their rates of lignin and humic substance conversion. Furthermore, lignin degradation in forest soil can rather slow down by interspecific competition than it is accelerated by cooperation of different microorganisms occupying specific nutritional niches. Therefore, the overall contribution of saprobic basidiomycetes depends on their particular eco-physiological status and the competitive pressure, and may be often lower than initially expected. Significant lignin transformation including partial mineralization is seemingly not exclusively dependent on exceptional high titres of ligninolytic enzymes but also on so far unknown factors. Higher endocellulase production and subsequent weight loss was found in microcosms where saprobic basidiomycetes were combined with indigenous microbes. Potentially, lignin degradation by the basidiomycetes may have increased cellulose availability to the indigenous microbes. [Copyright &y& Elsevier]

Abstract: The antimicrobial and preservative agent triclosan (TCS) is an emerging and persistent pollutant with a ubiquitous presence in the aquatic environment. Thus, TCS was treated with manganese peroxidase (MnP), laccase and the laccase-mediator system with 1-hydroxybenzotriazole. MnP was most effective in eliminating TCS among the three enzymatic treatments, with TCS concentration being reduced by about 94% after 30min following treatment with 0.5nkatmL−1 MnP and being almost completely eliminated after 60min. Furthermore, MnP (0.5nkatmL−1) caused the complete loss of bacterial growth inhibition by TCS after 30min and reduced the algal growth inhibition of TCS by 75% and 90% after 30 and 60min, respectively. These results strongly suggest that MnP is effective in removing the ecotoxicity of TCS. [Copyright &y& Elsevier]

Abstract: Delignification of corn stover by the white rot fungus Ceriporiopsis subvermispora in solid-state cultivation was evaluated for improving subsequent enzymatic hydrolysis. The results showed that C. subvermispora selectively degraded lignin by as much as 39.2% while the cellulose loss was less than 5% during 42d of cultivation. However, hemicellulose loss of up to 27.0% was concomitant with the lignin degradation. Ligninolytic enzymes, MnP and laccase, were detected during degradation of corn stover by C. subvermispora. For major hydrolytic enzymes, xylanase was the only enzyme detected. The enzymatic hydrolysis yield of corn stover delignified by C. subvermispora was remarkably improved, reaching about 57–67% overall glucose yield after 18–42d of pretreatment, while the glucose yield of the untreated was only 22%. A higher overall glucose yield of about 72% was obtained when the enzymatic hydrolysis of the 18d treated corn stover was supplemented with an accessory xylanase/cellulase enzyme complex. [Copyright &y& Elsevier]

Abstract: The ligninolytic machinery of the widely used model fungus Ceriporiopsis subvermispora includes the enzymes manganese-peroxidase (MnP) and laccase (Lcs). In this work the effect of Mn(II) on the secretion of MnP was studied. Cultures grown in the absence of Mn(II) showed high levels of mnp transcripts. However, almost no MnP enzyme was detected in the extracellular medium, either by enzymatic activity assays or Western blot hybridizations. In the corresponding mycelia, immuno-electron microscopy experiments showed high levels of MnP enzyme within intracellular compartments. These results suggest that in addition to its well-known effect on transcription regulation of mnp genes, manganese influences secretion of MnP to the extracellular medium. Experiments carried out in the presence of cycloheximide confirmed that the metal is required to secrete MnP already synthesized and retained within the cell. [Copyright &y& Elsevier]

Abstract: Interspecific fungal antagonism leads to biochemical changes in competing mycelia, including up-regulation of oxidative enzymes. Laccase, manganese peroxidase (MnP), manganese-repressed peroxidase (MRP) and lignin peroxidase (LiP) gene expression and enzyme activity were compared during agar interactions between Trametes versicolor and five other wood decay fungi resulting in a range of interaction outcomes from deadlock to replacement of one fungus by another. Increased laccase and Mn-oxidising activities were detected at all interaction zones, but there were few changes in activity in regions away from the interaction zone in T. versicolor mycelia compared to self-pairings. Whilst no LiP activity was detected in any pairing, low level LiP gene expression was detected. MnP activity was detected but not expression of MnP genes; instead, MRP could explain the observed activity. No relationship was found between extent of enzyme activity increase and interaction outcome. Similarities between patterns of gene expression and enzyme activity are discussed. [Copyright &y& Elsevier]

Summary: Copper (Cu) is an essential element for the physiological function of organisms. In basidiomycetes, Cu is necessary for the production of phenol oxidase enzymes such as laccase and tyrosinase. We isolated and characterized two genes, CcCTR1 and -2, from the model basidiomycete Coprinopsis cinerea. CcCTR1 and -2 showed similarity to the Cu transporter CTR1 in Saccharomyces cerevisiae. Both CcCTRs had a MLxxM motif that is conserved in other CTR homologs. The addition of Cu to a liquid culture of C. cinerea decreased the mRNA accumulation of CcCTR1 and -2. Heterologous expression of CcCTR1 in S. cerevisiae increased Cu sensitivity, suggesting that CcCTR1 is a Cu uptake transporter. Together, these results suggest that CcCTR1 plays an important role in Cu accumulation in C. cinerea. [Copyright &y& Elsevier]

Laccases are blue multicopper oxidases that catalyse the four-electron reduction of O2 to water coupled with the oxidation of small organic substrates. Secreted basidiomycete white-rot fungal laccases orchestrate this with high thermodynamic efficiency, making these enzymes excellent candidates for exploitation as industrial oxidants. However, these fungi are less tractable genetically than the ascomycetes, which predominantly produce lower-potential laccases. We address the state-of-play regarding expression of high reduction potential laccases in heterologous hosts, and issues regarding enzyme glycosylation status. We describe the synergistic role of structural biology, particularly in unmasking structure–function relationships following genetic modification and their collective impact on laccase yields. Such recent research draws closer the prospect of industrial quantities of designer, fit-for-purpose laccases. [Copyright &y& Elsevier]

Abstract  For the first time the dependence of completeness of pyrene degradation by the white-rot fungus Pleurotus ostreatus D1 on cultivation conditions was found. In Kirk’s medium about 65.6 ± 0.9% of the initial pyrene was metabolized after 3 weeks, with pyrene-4,5-dihydrodiol accumulating. This process was accompanied by laccase production only. In basidiomycetes rich medium, P. ostreatus D1 metabolized up to 89.8 ± 2.3% of pyrene within 3 weeks without pyrene-4,5-dihydrodiol accumulation throughout the time of cultivation. Phenanthrene and phthalic acid were identified as the metabolites produced from pyrene degradation under these conditions. Accumulation of phenanthrene with its subsequent disappearance was observed. One more metabolite probably was the product of phenanthrene degradation. Pyrene metabolism in basidiomycetes rich medium was accompanied first by laccase and tyrosinase production and later by versatile peroxidase production. The cell-associated activities of laccase, tyrosinase, and versatile peroxidase were found. The data obtained indicate that both enzymes (laccase and versatile peroxidase) are necessary for complete degradation of pyrene. Furthermore, both cell-associated and extracellular laccases can catalyse the first stages of pyrene degradation, and versatile peroxidase can be necessary for oxidation of the resulting metabolites. [ABSTRACT FROM AUTHOR]

Abstract: A comparison of laccase sequences highlighted the presence of a C-terminal extension of sixteen amino acids in POXA1b laccase – that represents the most thermostable isoenzyme among Pleurotus ostreatus laccases and exhibits a notable stability at alkaline pH (t 1/2 at pH 10=30 days) – whereas this tail is missing in the other analysed laccases from basidiomycetes. Site-directed mutagenesis experiments allowed us to demonstrate a role of the C-terminal tail of POXA1b in affecting its catalytic and stability properties. The truncated mutants lose the high stability at pH 10, while they show an increased stability at pH 5. The effect of substituting the residue Asp205 of POXA1b with an arginine was also analysed in the mutant POXA1bD205R. Following the mutation POXA1bD205R, a remarkable worsening of catalytic properties along with a decrease of substrate affinity and of enzyme stability were found. It was demonstrated that introducing Arg205 mutation in a highly conserved region perturbs the structural local environment in POXA1b, leading to a large rearrangement of the enzyme structure. Hence, a single substitution in the binding site introduces a local conformational change that not only leads to very different catalytic properties, but can also significantly destabilize the protein. [Copyright &y& Elsevier]

Abstract: This review integrates recent literature and our own data on the physiology of laccase and manganese peroxidase synthesis, focusing on the common characteristics and unique properties of individual fungi as well as on several approaches providing enhanced enzyme secretion. Firstly, the enzyme yield is species-dependent and strain-dependent and selection of new organisms with tremendous synthesis of these enzymes is possible. For example, in screening program the laccase activity of tested basidiomycetes varied from 0.5Uml−1 to 75Uml−1. Secondly, the carbon source and lignocellulosic substrate play a crucial role in enzyme production. Thus, laccase activity of Pseudotrametes gibbosa varied from 0.3Uml−1 (Avicel) to 13.7Uml−1 (lactose), while the substitution of wheat bran with walnut pericarp increased Cerrena unicolor manganese peroxidase yield from 0.7Uml−1 to 8.3Uml−1. Thirdly, aromatic compounds regulate the ligninolytic enzyme synthesis although their effect is very specific depending on fungi physiological peculiarities. 2,4,6-trinitrotoluene (TNT) supplemented to the medium at appropriate concentration significantly accelerated C. unicolor laccase production and 4-fold increased laccase specific activity. Fourthly, co-cultivation of appropriate fungi shows considerable promise as a strategy to highly enhance the enzyme production. For example, pairing of C. unicolor and Phellinus robustus 2-fold increased the total laccase yield. [Copyright &y& Elsevier]

A homogenous enzyme with both bilirubin oxidase and laccase activities was isolated from a submerged culture of the basidiomycete Pleurotus ostreatus mycelium and characterized. The yield of the enzyme was 127 μg/g dry biomass of the mycelium. The specific activity of the enzyme was 21 and 261 U/mg to bilirubin and to a laccase substrate ABTS, respectively. The intracellular phenol oxidase from the P. ostreatus mycelium was identified as bilirubin oxidase with the amino acid sequence highly homologous to that of the pox2 gene-encoded product. The enzyme displayed the maximal laccase activity at 50–55°C to all substrates examined, whereas the pH optimum was substrate-dependent and changed from 3.0 for ABTS to 7.0 for syringaldazine and guaiacol. The enzyme maintained catalytic activity within a broad pH range but was inactivated at pH 4.0. The enzyme was thermostable but very sensitive to metal chelating inhibitors. Trypan Blue (5 mg/liter) was completely decolorizated upon 3 h of incubation with the bilirubin oxidase (20 mU/ml) at room temperature. [ABSTRACT FROM AUTHOR]

Fungal peroxidases and phenoloxidases are widely used in aromatic toxic compounds degradation. Peroxidases, such as lignin peroxidase and manganese peroxidase, as well as laccases are mainly produced by basidiomycetes and to a lower extent by other fungi, such as ascomycetes. Peroxidase-encoding genes have been described and homologous expression has been achieved in basidiomycetes. Heterologous expression has also been achieved in some non-producing peroxidase ascomycetes, like Penicillium and Aspergillus. In this work, heterologous expression of peroxidase-encoding genes, lignin peroxidase, and manganese peroxidase was achieved in a zygomycete producing only phenoloxidases ( Amylomyces rouxii), aimed at coupling two different pathways used in nature for PCP removal in only one microbial strain. The ability of PCP removal was assayed with one of the obtained transformants, resulting in increased activity with respect to the ability of the parental strain cultured free of the inducer tyrosine (95% and 45%, respectively, of the initial PCP (12.5 mg L−1) in 120 h, or 100% and 49%, respectively, of the initial PCP after 144 h of liquid culture). [ABSTRACT FROM AUTHOR]

Abstract: Temporal fluctuations of present and expressed fungal genes encoding the ligninolytic enzyme laccase were examined bimonthly between March 2004 and April 2005 in the organic horizon of a beech forest Cambisol. Using two sets of degenerate primer pairs, we detected 73 different basidiomycete laccase genes from soil DNA extracts and 42 different transcripts of asco- and basidiomycetes from soil RNA extracts (cDNA). Phylogenetic analysis related the sequences to fungal taxa. The highest fungal laccase gene richness in soil DNA and RNA samples were found in August, October and January, and followed the input of fresh litter into soil. The highest change of the fungal laccase gene population was observed from October to January, but no distinct temporal change in the total soil laccase activity was measured. Present and expressed laccase gene populations were highly different, implying different subsets amplified with our primer sets and likely impacting future research strategies. Despite considerable variations in gene presence and expression, we found a steady expression and high soil enzyme activity of fungal laccases at each sampling date, thus presumably laccases have major impacts to soil organic matter turnover and stabilization processes. [Copyright &y& Elsevier]

A consortium of three basidiomycetes isolated from compost was investigated for pyrene degradation in soil microcosms. Pyrene concentration, glucose and ammonium evolution, moisture content, ligninolytic enzyme activities and phytotoxicity (germination index) on Lepidium sativum L. seeds were monitored. The fungal consortium grown on straw was found able to efficiently colonize soil and remove about 56 out of 100mgkg−1 of soil dry weight of pyrene in 28 days; in the meantime the germination index increased indicating a reduction of phytotoxicity. A glucose supply after 2 weeks was found useful to ensure fungal growth and activity; maintenance of moisture content below 70% allowed a good aeration of the system and improved degradation rates. Enzymatic assays showed that laccase and manganese independent peroxidase activity could have played a role in the degradation process. [Copyright &y& Elsevier]

• In forest soils, ectomycorrhizal and saprotrophic Agaricales differ in their strategies for carbon acquisition, but share common gene families encoding multi-copper oxidases (MCOs). These enzymes are involved in the oxidation of a variety of soil organic compounds. • The MCO gene family of the ectomycorrhizal fungus Laccaria bicolor is composed of 11 genes divided into two distinct subfamilies corresponding to laccases ( lcc) sensu stricto ( lcc1 to lcc9), sharing a high sequence homology with the coprophilic Coprinopsis cinerea laccase genes, and to ferroxidases ( lcc10 and lcc11) that are not present in C. cinerea. The fet3-like ferroxidase genes lcc10 and lcc11 in L. bicolor are each arranged in a mirrored tandem orientation with an ftr gene coding for an iron permease. Unlike C. cinerea, L. bicolor has no sid1/ sidA gene for siderophore biosynthesis. • Transcript profiling using whole-genome expression arrays and quantitative reverse transcriptase–polymerase chain reaction (qRT-PCR) revealed that some transcripts were very abundant in ectomycorrhizas ( lcc3 and lcc8), in fruiting bodies ( lcc7) or in the free-living mycelium grown on agar medium ( lcc9 and lcc10), suggesting a specific function of these MCOs. • The amino acid composition of the MCO substrate binding sites suggests that L. bicolor MCOs interact with substrates different from those of saprotrophic fungi. [ABSTRACT FROM AUTHOR]

Repeated prescribed burning alters the biologically labile fraction of nutrients and carbon of soil organic matter (SOM). Using a long-term (30 years) repeated burning experiment where burning has been carried out at a 2- or 4-year frequency, we analysed the effect of prescribed burning on gross potential C turnover rates and phenol oxidase activity in relation to shifts in SOM composition as observed using Fourier-transform infrared spectroscopy. In tandem, we assessed the genetic diversity of basidiomycete laccases. While the overall effect of burning was a decline in phenol oxidase activity, Shannon diversity and evenness of laccases was significantly higher in burned sites. Co-correspondence analysis of SOM composition and laccase operational taxonomic unit frequency data also suggested a strong correlation. While this correlation could indicate that the observed increase in laccase genetic diversity due to burning is due to increased resource diversity, a temporal replacement of the most abundant members of the assembly by an otherwise dormant pool of fungi cannot be excluded. As such, our results fit the intermediate disturbance hypothesis. Effects were stronger in plots burned in 2-year rotations, suggesting that the 4-year burn frequency may be a more sustainable practice to ensure the long-term stability of C cycling in such ecosystems. [ABSTRACT FROM AUTHOR]

The production of laccase by an indigenous strain of Pleurotus ostreatus HP-1 was studied on solid state fermentation. Culture parameters such as type and concentration of substrate, inoculum size, moisture content, pH, surfactant presence, temperature, and nitrogen source were optimized by conventional "one factor at a time" methodology. A maximum laccase yield of 3952 U g-1 of dry substrate optimized was obtained with wheat straw as substrate with five agar plugs as the inoculum, 60% moisture content, pH 5.0, surfactant concentration 0.015 gl-1, and nitrogen source (combination of L-asparagine and NH4NO3 at 10 mM concentration each) at incubation temperature 28°C. Enhancement in laccase activity was achieved with the use of various aromatic inducers and copper sulphate. Highest laccase activity of 14189 U g-1 of dry substrate was achieved using 0.28 mM copper sulphate under optimized conditions. Thus, the indigenous isolate seems to be a potential producer of laccase using SSF and can be exploited for further biotechnological applications. The process also promises economic utilization and value addition of agro-residues. [ABSTRACT FROM AUTHOR]

The present study is the first describing the sequencing of a fragment of the copper-oxidase domain of a laccase gene in the family Botryosphaeriaceae. The aim of this work was to assess the degree of genetic and evolutionary relationships of a laccase gene from Botryosphaeria rhodina MAMB-05 with other ascomycete and basidiomycete laccase genes. The 193-amino acid sequences of the copper-oxidase domain from several different fungi, insects, a plant, and a bacterial species were retrieved from GenBank and aligned. Phylogenetic analyses were performed using neighbor-joining, maximum parsimony, and Bayesian inference methods. The organisms studied clustered into five gene clades: fungi (ascomycetes and basidiomycetes), insects, plants, and bacteria. Also, the topologies showed that fungal laccases of the ascomycetes and basidiomycetes are clearly separated into two distinct clusters. This evidence indicated that B. rhodina MAMB-05 and other closely related ascomycetes are a new biological resource given the biotechnological potential of their laccase genes. [ABSTRACT FROM AUTHOR]

A new method for synthesis of the conductive complex between polyaniline ( PANI) and poly(2-acrylamido-2-methyl-1-propanosulfonic acid) ( PAMPS) was proposed; in this method, the immobilized laccase from the basidiomycete Trametes hirsuta is used as a biocatalyst for aniline oxidative polymerization. The conditions for laccase immobilization on CM cellulose by bifunctional Woodward’s reagent were optimized. The catalytic properties of immobilized and native laccases were compared. The immobilized laccase appeared an efficient catalyst for the oxidative radical polymerization of aniline on polysulfonic acid matrix at 4°C. It was demonstrated that the immobilized enzyme could be repeatedly used for enzymatic synthesis of this polymer. Several spectral characteristics of the PANI/PAMPS complexes synthesized at various pH values were studied. The conductance of PANI specimens produced using immobilized laccase as a catalyst was 13 mS/cm. [ABSTRACT FROM AUTHOR]

Anthropogenic nitrogen (N) deposition affects a wide range of soil processes including phenol oxidase (PO) activity and soil organic matter dynamics. Depression of phenol oxidase activity in response to N saturation is believed to be mediated by the activity of white-rot basidiomycetes, whose production of extracellular oxidative enzymes can be limited by high N availability. We examined the effect of short-term N deposition on basidiomycete laccase gene diversity and relative abundance in temperate oak forest soil in which significant decreases in phenol oxidase and increased SOM have been recorded in response to experimental N deposition. UniFrac was used to compare the composition of laccase genes between three control- and three nitrogen-fertilized (80 kg−1 ha−1 per year) oak forest soils. The relative abundance of laccase genes was determined from qPCR analysis of laccase and basidiomycete ITS gene abundances. Our results indicate that there was no significant shift in the composition of laccase genes between control- and N-fertilized soils, nor was there a significant change in the relative abundance of laccase genes. These data suggest that N deposition effects on mineral soil PO activity do not result from changes in laccase gene diversity of white-rot basidiomycetes but are likely the result of altered microbial abundance or expression in this ecosystem type. Furthermore, laccase gene composition may be tied to factors that structure microbial communities in general, as soil laccase gene communities are more similar to other forest soils than with the corresponding litter. [ABSTRACT FROM AUTHOR]

Aims: To select Trichoderma strains for enhanced laccase production in Pleurotus ostreatus or Agaricus bisporus cultures. Methods and Results: Laccase production by P. ostreatus and A. bisporus was evaluated in liquid (axenic) and solid (dual cultures) malt extract medium. Oxidation of ABTS, DMP and syringaldazine was evaluated in order to assess the potential of Trichoderma strains to enhance laccase production by basidiomycetes. Selected Pleurotus–Trichoderma interactions yielded higher increases in laccase volumetric activity and an additional laccase isoform was produced. By contrast, Agaricus–Trichoderma interactions lead to smaller increases on laccase volumetric activity, probably as result of repression (or degradation) towards one of the laccases isoforms. Conclusions: The strains of P. ostreatus and A. bisporus assessed in this work showed good potential as laccase producers. The Trichoderma-mediated biological stimulation of laccase production by P. ostreatus and A. bisporus is relevant in order to develop highly productive processes. Significance and Impact of the Study: Extracellular laccases from basidiomycetes are produced only in small amounts. It is therefore important to increase process productivity for potential industrial applications. The results from this study enable the selection Trichoderma strains capable of increasing laccase production by P. ostreatus or A. bisporus in dual cultures. [ABSTRACT FROM AUTHOR]

Aims: To isolate and characterize the laccase isoforms from S. ochraceum 1833 – a new active producer of high extracellular laccase activity. Methods and Results: Three laccase isoforms (laccases I, II and III) with 57·5, 59·5 and 63 kDa molecular masses respectively were purified from S. ochraceum 1833 and in contrast to the known laccases had strongly pronounced absorption at 611 nm with molar extinction coefficients ranging from 7170 to 7830 mol−1 l cm−1. All isoforms showed maximal activity with ABTS at low pH (≤2) and temperatures in the range 70–80°C, were stable for long time of incubation at high temperature (60–80°C) and at pH values ranging from 2 to 6. Laccase II showed a higher activity and wider substrate specificity. N-terminal amino acid sequence analysis of the purified laccase II (VQIGPVTDLH) showed 80% identity with the N-terminal amino acid sequence of laccase from Lentinula edodes [ Appl Microbiol Biotechnol 60 (2002) 327]. Conclusions: Elevated temperature optima, high thermo- and pH-stabilities, the broad substrate specificity of the isoforms make the laccases from S. ochraceum 1833 a suitable model for biotechnological processes proceeding at high temperatures. Significance and Impact of the Study: For the first time, new basidiomycete strain S. ochraceum was reported as a producer of novel thermostable, pH stable, acidophilic laccases with unusual spectral properties. [ABSTRACT FROM AUTHOR]

The basidiomycete Lentinus tigrinus was cultured in media containing copper ions added at different growth stages. Copper ions at increased concentrations decelerated of the fungal biomass accumulation. The later Cu2+ ions were added, the better the fungal mycelium developed, and the toxic effect of Cu2+ was less pronounced. The maximum laccase activity (47 U/ml) was observed in the presence of 1.5–2.0 mM Cu2+ added on day 4 of cultivation. [ABSTRACT FROM AUTHOR]

The exploration of seven physiologically different white rot fungi potential to produce cellulase, xylanase, laccase, and manganese peroxidase (MnP) showed that the enzyme yield and their ratio in enzyme preparations significantly depends on the fungus species, lignocellulosic growth substrate, and cultivation method. The fruit residues were appropriate growth substrates for the production of hydrolytic enzymes and laccase. The highest endoglucanase (111 U ml−1) and xylanase (135 U ml−1) activities were revealed in submerged fermentation (SF) of banana peels by Pycnoporus coccineus. In the same cultivation conditions Cerrena maxima accumulated the highest level of laccase activity (7,620 U l−1). The lignified materials (wheat straw and tree leaves) appeared to be appropriate for the MnP secretion by majority basidiomycetes. With few exceptions, SF favored to hydrolases and laccase production by fungi tested whereas SSF was appropriate for the MnP accumulation. Thus, the Coriolopsis polyzona hydrolases activity increased more than threefold, while laccase yield increased 15-fold when tree leaves were undergone to SF instead SSF. The supplementation of nitrogen to the control medium seemed to have a negative effect on all enzyme production in SSF of wheat straw and tree leaves by Pleurotus ostreatus. In SF peptone and ammonium containing salts significantly increased C. polyzona and Trametes versicolor hydrolases and laccase yields. However, in most cases the supplementation of media with additional nitrogen lowered the fungi specific enzyme activities. Especially strong repression of T. versicolor MnP production was revealed. [ABSTRACT FROM AUTHOR]

The white-rot fungus Lentinus edodes produced d-melibiose-specific lectins and two laccase forms in a lignin-containing medium. The maxima of laccase and lectin activities coincided, falling within the period of active mycelial growth. The enzymes and lectins were isolated and purified by gel filtration followed by anion-exchange chromatography. The L. edodes lectins were found to be able to stabilize the activity of the fungus’s own laccases. Lectin activity during the formation of lectin–enzyme complexes remained unchanged. [ABSTRACT FROM AUTHOR]

The potential of a consortium of three basidiomycete mycelia isolated from compost to degrade polycyclic aromatic hydrocarbons (PAH) was first evaluated using a test based on decolorization of Poly R-478 dye. When pre-grown on straw, the consortium decolorized the dye by 83% in 7 days and generated a laccase activity of 663IUl−1. Its ability to degrade naphthalene was investigated in soil microcosms specially suited for this volatile PAH. The kinetic study was conducted at a maximal naphthalene concentration of 500mgkg−1 of soil. Naphthalene concentration, CO2 evolution and phytotoxicity (germination index, GI%) on Lepidium sativum seeds were monitored. The naphthalene concentration decreased by about 70% in three weeks in the presence of metabolic activity, while the GI% increased indicating reduced phytotoxicity. [Copyright &y& Elsevier]

Abstract: Activity and production of extracellular enzymes by saprotrophic litter-decomposing basidiomycetes Hypholoma fasciculare and Rhodocollybia butyracea was studied in microcosms with reconstructed L, O and Ah horizons of a soil profile of Quercus petraea forest soil. Both H. fasciculare and R. butyracea colonized the L layer of microcosms rapidly, while the colonization of O layer was slower. The Ah layer was substantially colonized only by R. butyracea. Enzyme activities in the soil microcosms decreased from the L layer>O layer>Ah layer and activities in microcosms inoculated with the fungi were quite similar to each other. Compared to control, the most apparent was the increase of ligninolytic enzyme activities. Laccase activities in H. fasciculare and R. butyracea-colonized L layers were 3-fold compared to control and the activity maxima of Mn-peroxidase in fungus-colonized O layers were 2–3-fold and in the L layers up to 40-fold compared to controls. Activities of cellulolytic enzymes, chitinase and acidic phosphatase in both fungal treatments were higher in the L layer on weeks 2–6 while the activity of alkaline phosphatase did not show differences between fungus-colonized and control treatments. Both fungi decreased fungal CFU in the L layer but significantly increased the counts in the O layer. Both fungi also increased bacterial CFU in the O layer, R. butyracea more than H. fasciculare. The analysis of fungal and bacterial biomass based on ergosterol content and PLFA analysis showed a sharp decrease from L to Ah layer, but did not show significant differences among treatments. [Copyright &y& Elsevier]

A white-rot basidiomycete, Phlebia tremellosa, produced a laccase that showed increased activity during degradation of phthalates. A laccase was purified through the ion exchange chromatography and preparative gel electrophoresis, and the estimated molecular weight was 75 kDa. The optimum pH and temperature of the purified laccase was pH 4.0 and 20 °C, respectively. The Km value of the enzyme was 55.7 μM, and the Vmax was 0.0541 OD min−1 U−1 for o-tolidine. Purified laccase reduced the estrogenic activity of four different endocrine-disrupting chemicals. However, this effect was reduced by a laccase inhibitor, kojic acid, which confirmed that the laccase was involved in the removal of estrogenic activity. [ABSTRACT FROM AUTHOR]

It has been shown that the fungus Lentinus edodes grown on a solid wort agar substrate produces intracellular enzymes, including Mn-dependent peroxidase, laccase, and tyrosinase as a family of isoforms. The composition of the complex (containing one to four forms of each enzyme) varied during the basidiomycete life cycle. The activity of oxidases was maximal at the stage of nonpigmented mycelium and at the stages of a brown mycelial mat and a fruit body. The activity of tyrosinase increased in the course of mycelium pigmentation and had two maxima: at the stage of a brown mycelial mat and at the stage of a fruit body. Laccase and tyrosinase activities were shown to increase sharply upon addition of oak sawdust extract to the culture medium as compared with the enzyme activities of mycelium grown on wort agar alone. It was established that the effect of phenol oxidase substrates on the growing mycelium consists in a twofold acceleration of the process of morphogenesis in the fungus L. edodes. [ABSTRACT FROM AUTHOR]

Abstract: Cyathus bulleri, a ligninolytic fungus, produces a single laccase the internal peptides (3) of which bear similarity to laccases of several white rot fungi. Comparison of the total amino acid composition of this laccase with several fungal laccases indicated dissimilarity in the proportion of some basic and hydrophobic amino acids. Analysis of the circular dichroism spectrum of the protein indicated 37% α-helical, 26% β-sheet and 38% random coil content which differed significantly from that in the solved structures of other laccases, which contain higher β-sheet structures. The critical role of the carboxylic group containing amino acids was demonstrated by determining the kinetic parameters at different pH and this was confirmed by the observation that a critical Asp is strongly conserved in both Ascomycete and Basidiomycete laccases. The enzyme was denatured in the presence of a number of denaturing agents and refolded back to functional state with copper. In the folding experiments under alkaline conditions, zinc could replace copper in restoring 100% of laccase activity indicating the non-essential role of copper in this laccase. The laccase was expressed in Escherichia coli by a modification of the ligation-anchored PCR approach making it the first fungal laccase to be expressed in a bacterial host. The laccase sequence was confirmed by way of analysis of a 435 bp sequence of the insert. [Copyright &y& Elsevier]

Abstract: Lentinus edodes and Pleurotus species from various origins were compared for the first time for their ability to produce lignocellulolytic enzyme in solid-state (SSF) and submerged (SF) fermentation of various plant raw material. Fungi cultivation in identical culture conditions revealed wide differences among both species and strains of the same species. The yields of CMCase (62.3Uml−1), xylanase (84.1Uml−1), FPA (5.9Uml−1), and laccase (4103Ul−1) are the best so far obtained with the strains of oyster mushrooms. The study pointed out that the nature of lignocellulosic material and the method of fungi cultivation are factors determining the expression of lignocellulolytic potential of fungi as well as the ratio of individual enzymes in enzyme complex. SSF of tree leaves is favorable for laccase and MnP secretion by the majority L. edodes and Pleurotus strains, whereas SF provides better production of hydrolytic enzymes. [Copyright &y& Elsevier]

The decolorization of industrial dyes was investigated using extracellular enzymes produced by 21 basidiomycetes, mainly edible mushrooms. Among the 27 dyes used in this study, nine were decolorized by over 40%. Most fungi decolorized Acid Orange 20, but they showed different specificities in the case of the other dyes. Determination of activity staining by native polyacrylamide gel electrophoresis revealed that all the decolorization activities corresponded to 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) oxidation activities. [Copyright &y& Elsevier]

Although field studies have demonstrated an ecosystem-specific effect of experimental atmospheric nitrogen (N) deposition on litter decomposition, a mechanistic understanding of how ligninolytic microbial communities respond to atmospheric deposition is lacking. Because high levels of inorganic N suppress lignin decomposition by some basidiomycetes, it is plausible that the abundance and activity of these key microorganisms underlies differential ecosystem responses of decomposition to atmospheric N deposition. We hypothesize that: (a) atmospheric N deposition will cause an ecosystem-specific reduction in basidiomycete activity and abundance with greatest decreases in ecosystems with lignin-rich forest litter and (b) the abundance of lignin degrading basidiomycetes will be positively correlated with ligninolytic enzyme activity. To test these hypotheses, we measured the effects of experimental N deposition on the potential activity of phenol oxidase enzymes, and the abundance of basidiomycete genes encoding laccase, a primary phenol oxidase enzyme, in three hardwood forests spanning a range of leaf litter lignin content. The black oak-white oak (BOWO) contains high lignin litter, the sugar maple-basswood (SMBW) has low lignin litter, and the sugar maple-red oak (SMRO) is intermediate. An ecosystem by N deposition interaction significantly influenced phenol oxidase activity in the surface soil ( P = 0.05), where phenol oxidase activity decreased with increasing experimental N deposition in the BOWO ecosystem. No consistent response to N deposition was evident for surface soil phenol oxidase activity within either the SMRO or SMBW ecosystem. This interaction did not influence laccase gene abundance. Instead, basidiomycete laccase gene abundance was reduced by experimental N deposition (main effect) in surface soil. There was only a weak correlation between basidiomycete laccase gene abundance and potential phenol oxidase enzyme activity, suggesting that the abundance of organisms possessing laccase genes may not control phenol oxidase activity in soil. Our results suggest that the regulation of laccase gene expression may mediate the decomposition response to atmospheric N deposition. [ABSTRACT FROM AUTHOR]