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C4 grasses are common species in rangelands around the world and represent an attractive option for second-generation biofuel production. Although they display high polysaccharide content and reach great levels of biomass accumulation, there is a major technical issue to be addressed before they can be used for bioethanol industrial production: lignin removal. Concerning this, Pycnoporus and Ganoderma fungal genera have been highlighted due to their ability to hydrolyze lignocellulose in biological pretreatments. Our goals here were to evaluate the pretreatment efficiency using the secretome of species from Pycnoporus and Ganoderma spp. harvested from a glucose-free inductive medium (using a C4 grass) and to identify the fungal enzymatic activities responsible for the lignin degradation and glucose release. Our results show that P. sanguineus secretome exhibits a higher activity of lignocellulolytic enzymes such as cellulases, xylanases, laccases, and manganese peroxidases compared with that from G. resinaceum. Interestingly, zymograms in the presence of 2 M glucose suggest that a β-glucosidase isoform from P. sanguineus could be glucose tolerant. The proteomic approach carried out allowed the identification of 73 and 180 different proteins in G. resinaceum and P. sanguineus secretomes, respectively, which were functionally classified in five main categories and a miscellaneous group. These results open new avenues for future experimental work that lead to a deeper comprehension and a greater application of the mechanisms underlying lignocellulosic biomass degradation.

Woody biomass represents an important source of carbon on earth, and its global recycling is highly dependent on Agaricomycetes fungi. White-rot Basidiomycetes are a very important group in this regard, as they possess a large and diverse enzymatic repertoire for biomass decomposition. Among these enzymes, the recently discovered lytic polysaccharide monooxygenases (LPMOs) have revolutionized biomass processing with their novel oxidative mechanism of action. The strikingly high representation of LPMOs in fungal genomes raises the question of their functional versatility. In this work, we studied an AA9 LPMO from the white-rot basidiomycete Pycnoporus sanguineus, PsAA9A. Successfully produced as a recombinant secreted protein in Pichia pastoris, PsAA9A was found to be a C1-specific LPMO active on cellulosic substrates, generating native and oxidized cello-oligosaccharides in the presence of an external electron donor. PsAA9A boosted cellulolytic activity of glysoside hydrolases from families GH1, GH5, and GH6.This study serves as a starting point towards understanding the functional versatility and biotechnological potential of this enzymatic family, highly represented in wood decay fungi, in Pycnoporus genus. KEY POINTS: • PsAA9A is the first AA9 from P. sanguineus to be characterized. • PsAA9A has activity on cellulose, producing C1-oxidized cello-oligosaccharides. • Boosting activity with GH1, GH5, and GH6 was proven.

Carboxylic acid reductases (CARs) are attracting burgeoning attention as biocatalysts for organic synthesis of aldehydes and their follow-up products from economic carboxylic acid precursors. The CAR enzyme class as a whole, however, is still poorly understood. To date, relatively few CAR sequences have been reported, especially from fungal sources. Here, we sought to increase the diversity of the CAR enzyme class. Six new CAR sequences from the white-rot fungus Pycnoporus cinnabarinus were identified from genome-wide mining. Genome and gene clustering analysis suggests that these PcCAR enzymes play different natural roles in Basidiomycete systems, compared to their type II Ascomycete counterparts. The cDNA sequences of all six Pccar genes were deduced and analysis of their corresponding amino acid sequence showed that they encode for proteins of similar properties that possess a conserved modular functional tri-domain arrangement. Phylogenetic analyses showed that all PcCAR enzymes cluster together with the other type IV CARs. One candidate, PcCAR4, was cloned and over-expressed recombinantly in Escherichia coli. Subsequent biotransformation-based screening with a panel of structurally-diverse carboxylic acid substrates suggest that PcCAR4 possessed a more pronounced substrate specificity compared to previously reported CARs, preferring to reduce sterically-rigid carboxylic acids such as benzoic acid. These findings thus present a new functionally-distinct member of the CAR enzyme class.

The degradation of 2,2′,4,4′-tetrabromodiphenyl ether (BDE-47) by Pycnoporus sanguineus was investigated in order to explore the impact of the heavy metal Cu2+ on BDE-47 decomposition and the subsequent formation of metabolites, as well as to further elucidate the degradation mechanism of BDE-47. An increase in degradation rate from 18.63% to 49.76% in the first four days and its stabilization at (51.26 ± 0.08)% in the following days of BDE-47 incubation were observed. The presence of Cu2+ at 1 and 2 mg/L was found to promote the degradation rate to 56.41% and 60.79%, respectively, whereas higher level of Cu2+ (≥ 5 mg/L) inhibited the removal of BDE-47. The similar concentration effects of Cu2+ was also found on contents of fungal protein and amounts of metabolites. Both intracellular and extracellular enzymes played certain roles in BDE-47 transportation with the best degradation rate at 27.90% and 27.67% on the fourth and third day, individually. During the degradation of BDE-47, four types of hydroxylated polybrominated diphenyl ethers (OH-PBDEs), i.e., 6′-OH-BDE-47, 5′-OH-BDE-47, 4′-OH-BDE-17, 2′-OH-BDE-28, and two bromophenols, i.e., 2,4-DBP and 4-BP were detected and considered as degradation products. These metabolites were further removed by P. sanguineus at rates of 22.42%, 23.01%, 27.04%, 27.96%, 64.21%, and 40.62%, respectively.

14 p.-8 fig.-1 tab., White-rot (WR) fungi are pivotal decomposers of dead organic matter in forest ecosystems and typically use a large array of hydrolytic and oxidative enzymes to deconstruct lignocellulose. However, the extent of lignin and cellulose degradation may vary between species and wood type. Here we combined comparative genomics,transcriptomics and secretome proteomics to identify conserved enzymatic signatures at the onset of wood decaying activity within the Basidiomycota genus Pycnoporus. We observed strong conservation in the genome structures and the repertoires of protein coding genes across the four Pycnoporus species described to date,despite the species having distinct geographic distributions. We further analyzed the early response of P. cinnabarinus, P. coccineus and P. sanguineus to diverse (ligno)-cellulosic substrates. We identified a conserved set of enzymes mobilized by the three species for breaking down cellulose, hemicellulose and pectin.The co-occurrence in the exo-proteomes of H2O2 producing enzymes with H2O2 consuming enzymes was a common feature of the three species, although each enzymatic partner displayed independent transcriptional regulation. Finally, cellobiose dehydrogenase-coding genes were systematically co-regulated with at least one AA9 LPMO gene, indicative of enzymatic synergy in vivo. This study highlights a conserved core white-rot fungal enzymatic mechanism behind the wood decaying process., This work was supported by The French National Agency for Research (ANR-14-CE06-0020). The work conducted by the U.S. Department of Energy Joint Genome Institute, a DOE Office of Science User Facility, is supported by the Office of Science of the U.S. Department of Energy under Contract No. DE-AC02-05CH11231. The work by CSIC was supported by the Ministerio de Ciencias, Innovación y Universidades de España (BIO2017-86559-R).

Biorecovery is emerging as a promising approach to retrieve gold from various sources, while its efficiency is usually restricted by the limited functional groups on natural microbial biomass surface. This study aims to intensify Pycnoporus sanguineus boosted sorption-reduction coupled gold biorecovery process via microbial surface modification. Results showed that grafting polyallylamine hydrochloride onto P. sanguineus biomass surface increased amino group content on microbial biomass surface from 1.29 to 2.81 mmol/g. When applying modified biomass to gold biorecovery with initial gold concentrations of 1.0, 2.0 and 3.0 mM, biosorption equilibrium time shortened to the 12.5%, 37.5% and 41.7% of those obtained with pristine biomass, and sorption rate constants correspondingly increased to 11.2, 3.1 and 3.7 folds as well. Maximum sorption capacity increased 30% and the affinity between biomass and gold enhanced heavily after microbial surface modification. Meanwhile, microbial surface modification favored gold reduction and gold nanoparticles (AuNPs) formation. The change of microbial biomass morphology from smooth surface with some branched structure to layered stacking structure with many pores and the increase of amino group content on microbial biomass surface were the main impetus for the gold bioreocovery process intensification.

Bioremediation is a strategy to mitigate environmental impacts of hazardous pollutants from anthropogenic sources. Natural byproducts, including agroindustrial wastes (AW) can be used to induce enzyme biosynthesis, leading up to enhancement of pollutants degradation process. Therefore, this study aimed to evaluate the use of cupuacu, Theobroma grandiflorum AW as Pycnoporus sanguineus Laccase (Lac) inducer in order to promote 17-α-ethinylestradiol (EE2) bioremediation. The macro and micro-nutrients levels of cupuacu AWs were evaluated in order to establish further correlations with enzymatic biosynthesis induction. The fungus was cultivated for 7 days in temperature of 28 ± 2 °C and agitation of 150 rpm. For bioremediation, Lac enzymatic extract was added to EE2 solution (10 µg mL−1) and the percentage of removal was evaluated by HPLC after 1–24 hr of reaction. At optimized conditions, the enzyme extract production was remarkably enhanced by adding only 1% (w/v) of cupuacu AW. Lac activity reached ...

Since fossil fuel resources are limited there is a necessity to produce alternative types of fuel that are renewable and eco-friendly. Basidiomycetes are potential sources of enzymes that can be used for biofuel production. The current study aimed to isolate basidiomycetes from Sri Lanka, screen them for lignocellulose degrading enzymes, namely cellulase, xylanase, laccase, Mn peroxidase and lignin peroxidase and study the effect of potential inducers of laccase production. Among the eighteen basidiomycetes isolated, Pycnoporus sp. produced the highest cellulase activity (0.23 FPU/ml) whereas Phlebiopsis sp. produced the highest xylanase activity (5.4 U/ml). Earliella scabrosa produced the highest laccase (91.2 U/l) and Mn peroxidase (17.5 U/l) activities. Lignin peroxidase activity was not detected from the isolates. Effect of alkali lignin, Cu2+ and rice bran, three potential inducers, on laccase production by E. scabrosa, Pycnoporus sp. and Trametes hirsuta (M40) was studied. Results indicated that alkali lignin (2 g/l) significantly increased laccase production from Pycnoporus sp. and T. hirsuta (M40) while Cu2+ increased laccase production from E. scabrosa and T. hirsuta at 200 μM. Use of rice bran (10 g/l) resulted in higher laccase production from E. scabrosa and Pycnoporus sp. High laccase activity (79600 U/l) was obtained from E. scabrosa by using 50 g/l of rice bran and by extending the incubation period to 18 days. The study concluded that some of the basidiomycetes isolated can produce significant lignocellulose degrading enzyme activities.

Mono and dual cultures of four white-rot basidiomycete species were evaluated for cellulase and xylanase activity under submerged fermentation conditions. Co-cultivation of Pycnoporus coccineus or Trametes hirsuta with Schizophyllum commune displayed antagonistic interactions resulting in the decrease of endoglucanase and total cellulase activities. In contrast, increases in cellulase and xylanase activity were revealed through the compatible interactions of Irpex lacteus with S. commune. Co-cultivation conditions were optimized for maximum enzyme production by I. lacteus and S. commune, the best producers of cellulase/xylanase and β-glucosidase, respectively. An optimized medium for the target enzyme production by the mixed culture was established in a laboratory fermenter yielding 7U/mL total cellulase, 142U/mL endoglucanase, 104U/mL xylanase, and 5.2U/mL β-glucosidase. The dual culture approach resulted in an enzymatic mixture with 11% improved lignocellulose saccharification potential compared to enzymes from a monoculture of I. lacteus.

White rot fungi have been proved to be a promising option for the removal of heavy metals, understanding their toxic response to heavy metals is conducive to developing and popularizing fungi-based remediation technologies so as to lessen the hazard of heavy metals. In this study, Cr(VI)-induced oxidative stress and apoptosis in Pycnoporus sanguineus, a species of white rot fungi were investigated. The results suggested that high level of Cr(VI) promoted the formation of ROS, including H 2 O 2 , O 2 •− and ·OH. With the increment of Cr(VI) concentration, the SOD and CAT activity along with GSH content increased within the first 24 h, but decreased afterward, companied with a significant enhancement of MDA content. Cr(VI)-induced oxidative damage further caused and aggravated apoptosis in P. sanguineus, especially at Cr(VI) concentrations above 20 mg/L. Cr(VI)-induced apoptosis was involved with mitochondrial dysfunction including mitochondrial depolarization, the enhancement of mitochondrial permeability and release of cytochrome c. The early and late apoptosis hallmarks, such as metacaspase activation, phosphatidylserine (PS) externalization, DNA fragmentation and the nuclear condensation and fragmentation were observed. Moreover, we also found disturbances of ion homeostasis, which was featured by K + effluxes and overload of cytoplasmic and mitochondrial Ca 2+ .Based on these results, we suggest that Cr(VI) induced oxidative stress and apoptosis in white rot fungi, P. sanguineus .

Pycnoporus sanguineus, an edible mushroom, produces antimicrobial and antitumor bioactive compounds and pH- and thermo- stable laccases that have multiple potential biotechnological applications. Here we reported the complete genome of the species Pycnoporus sanguineus ACCC 51,180 by using the combination of Illumina HiSeq X Ten and the PacBio sequencing technology. The represented genome is 36.6 Mb composed of 59 scaffolds with 12,086 functionally annotated protein-coding genes. The genome of Pycnoporus sanguineus encodes at least 19 biosynthetic gene clusters for secondary metabolites, including a terpene cluster for biosynthesis of the antitumor clavaric acid. Seven laccases were identified, while 22 genes were found to be involved in the kynurenine pathway in which the intermediate metabolite 3-hydroxyanthranilic acid were catalyzed by laccases into cinnabarinic acid. This study represented the third genome of the genus Pycnoporus, and wound facilitate the exploration of useful sources from Pycnoporus sanguineus for future industrial applications.

The influence of Cr(VI) on the degradation of tetrabromobisphenol A (TBBPA) by a typical species of white rot fungi, Pycnoporus sanguineus , was investigated in this study. The results showed that P. sanguineus together with its intracellular and extracellular enzyme could effectively degrade TBBPA. The degradation efficiency of TBBPA by both P. sanguineus and its enzymes decreased significantly when Cr(VI) concentration increased from 0 to 40 mg/L. The subsequent analysis about cellular distribution of TBBPA showed that the extracellular amount of TBBPA increased with the increment of Cr(VI) concentration, but the content of TBBPA inside fungal cells exhibited an opposite variation tendency. The inhibition of TBBPA degradation by P. sanguineus was partly attributed to the increase of cell membrane permeability and the decrease of cell membrane fluidity caused by Cr(VI). In addition, the decline of H + -ATPase and Mg 2+ -ATPase activities was also an important factor contributing to the suppression of TBBPA degradation in the system containing concomitant Cr(VI). Moreover, the activities of two typical extracellular lignin-degrading enzymes of P. sanguineus , MnP and Lac, were found to descend with ascended Cr(VI) level. Cr(VI) could also obviously suppress the gene expression of four intracellular enzymes implicated in TBBPA degradation, including two cytochrome P450s, glutathione S-transferases and pentachlorophenol 4-monooxygenase, which resulted in a decline of TBBPA degradation efficiency by fungal cells and intracellular enzyme in the presence of Cr(VI). Overall, this study provides new insights into the characteristics and mechanisms involved in TBBPA biodegradation by white rot fungi in an environment where heavy metals co-exist.

International audience; Production of second-generation bioethanol uses lignocellulose from agricultural by-products such as sugarcane bagasse (SCB). A lignocellulose pre-treatment is required to degrade lignin, ensuring further efficient sacchar-ification. Two experimental designs were set up to define culture conditions of Pycnoporus sanguineus in meso-cosms to increase laccase activities and thus delignification. The first experimental design tested the effect of phenolic complementation (via coffee pulp) and the use of urea as a simple nitrogen source and the second defined more precisely the percentages of coffee pulp and urea to enhance delignification. The responses measured were: lignocellulolytic activities, laccase isoform profiles by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and the chemical transformation of the substrate using solid-state NMR of 13C. Adding 10% of coffee pulp increased laccase activities and fungal biomass (32.5% and 16% respectively), enhanced two constitutive isoforms (R f 0.23 and 0.27), induced a new isoform (R f 0.19) and led to a decrease in total aromatics. However, higher concentrations of coffee pulp (25%) decreased laccase and cellulase activities but no decrease in aromaticity was observed, potentially due to the toxic effect of phenols from coffee pulp. Moreover, laccase production was still inhibited even for lower concentrations of urea (0-5%). Our findings revealed that an agricultural by-product like coffee pulp can enhance laccase activity-though to a threshold-and that urea limited this process, indicating that other N-sources should be tested for the biological delignification of SCB.

A laccase from the basidiomycete Pycnoporus sanguineus strain RVAN5 was evaluated for its ability to decolorize synthetic dyes and denim bleaching. Dye color reduction and denim bleaching were monitored at different dye concentrations and incubation times. Dye decolorization by Pycnoporus sanguineus fungal crude extract (FCE) ranged from 80 to 96% within 2-4 h at 25-65 °C. Comparable results were obtained when violuric acid (VA) was added as mediator to the FCE, however, the number of decolorized dyes increased significantly. Dye decolorization rates with VA varied of initial and final optical density (595 nm) values of 2.5-3.0 and 0.2-0.02, respectively. P. sanguineus FCE had no substantial effect on denim bleaching when used alone, notwithstanding, the mixture of FCE with VA (10 mM) showed significant denim color reduction values and considerably higher than those obtained with a bleaching enzyme from a commercial formulation; CIElab values obtained with FCE/VA mixture were of ΔL = 6.4, versus a ΔL 1.4 value obtained with an enzyme from commercial formulation.

The aim of this study was to investigate the production profile of Pleurotus albidus and Pycnoporus sanguineus on different waste substrates containing natural phenolics, and also to investigate whether phenolic-rich substrates can improve the phenolic content of these macrofungi. The medium formulated with Pinus sp. sawdust (PSW) made possible the highest yields (2.62±0.73%) of P. sanguineus. However, the supplementation of PSW with apple waste (AW) resulted in better P. albidus yields (23.94±2.92%). The results indicated that the substrate composition affected macrofungi production, also the chemical composition and the presence of phenolic compounds in the production media influence phenolic content and antioxidant activity in macrofungi.

Identification of bioactive components from complex natural product extracts can be a tedious process that aggravates the use of natural products in drug discovery campaigns.This study presents a new approach for screening antimicrobial potential of natural product extracts by employing a bioreporter assay amenable to HPLC-based activity profiling.A library of 116 crude extracts was prepared from fungal culture filtrates by liquid-liquid extraction with ethyl acetate, lyophilised, and screened against Escherichia coli using TLC bioautography. Active extracts were studied further with a broth microdilution assay, which was, however, too insensitive for identifying the active microfractions after HPLC separation. Therefore, an assay based on bioluminescent E. coli K-12 (pTetLux1) strain was coupled with HPLC micro-fractionation.Preliminary screening yielded six fungal extracts with potential antimicrobial activity. A crude extract from a culture filtrate of the wood-rotting fungus, Pycnoporus cinnabarinus (Jacq.) P. Karst. (Polyporaceae), was selected for evaluating the functionality of the bioreporter assay in HPLC-based activity profiling. In the bioreporter assay, the IC50 value for the crude extract was 0.10 mg/mL. By integrating the bioreporter assay with HPLC micro-fractionation, the antimicrobial activity was linked to LC-UV peak of a compound in the chromatogram of the extract. This compound was isolated and identified as a fungal pigment phlebiarubrone.HPLC-based activity profiling using the bioreporter-based approach is a valuable tool for identifying antimicrobial compound(s) from complex crude extracts, and offers improved sensitivity and speed compared with traditional antimicrobial assays, such as the turbidimetric measurement.

The optimization of ligninolytic enzyme (LE) activities in a novel fungal co-culture between Pycnoporus sanguineus and Beauveria brongniartii were studied using a Plackett-Burman experimental design (PBED) and a central composite design (CCD). In addition, H

In current study, we investigated the changes of proteome profiles of Pycnoporus sanguineus after a single exposure of Cr(VI), TBBPA and a combined exposure of TBBPA and Cr(VI), with the goal of illuminating the cellular mechanisms involved in the interactions of co-existed TBBPA and Cr(VI) with the cells of P. sanguineus at the protein level. The results revealed that some ATP-binding cassette (ABC) transporters were obviously induced by these pollutants to accelerate the transportation, transformation and detoxification of TBBPA and Cr(VI). Cr(VI) could inhibit the bioremoval of its organic co-pollutants TBBPA through suppressing the expression of several key proteins related to the metabolism of TBBPA by P. sanguineus, including two cytochrome P450s, pentachlorophenol 4-monooxygenase and glutathione S-transferases. Furthermore, Cr(VI) possibly reduced the cell vitality and growth of P. sanguineus by enhancing the expression of imidazole glycerol phosphate synthase as well as by decreasing the abundances of proteins associated with the intracellular metabolic processes, such as the tricarboxylic acid cycle, purine metabolism and glutathione biosynthesis, thereby adversely affecting the biotransformation of TBBPA. Cr(VI) also inhibited the expression of peptidyl prolyl cis/trans isomerases, thus causing the damage of cell membrane integrity. In addition, some important proteins participated in the resistance to Cr(VI) toxicity were observed to up-regulate, including heat shock proteins, 26S proteasome, peroxiredoxins and three critical proteins implicated in S-adenosyl methionine synthesis, which contributed to reducing the hazard of Cr(VI) to P. sanguineus. The results of this study provide novel insights into the physiological responses and molecular mechanism of white rot fungi P. sanguineus to the stress of concomitant TBBPA and Cr(VI).

The effects of Cu(2+) on the activity and expression of laccase were investigated in seven different strains of Pycnoporus coccineus collected from different regions in Korea. Cu(2+) was toxic to mycelial growth at concentrations greater than 0.5 mM CuSO4 and showed complete growth inhibition at 1 mM in the liquid culture. However, Cu(2+) significantly upregulated the extracellular laccase activity at 0.2 mM in five strains of P. coccineus, IUM4209, IUM0032, IUM0450, IUM0470, and IUM4093, whereas two strains, IUM0253 and IUM0049, did not respond to Cu(2+), despite being closely related to the other five strains. Subsequent RT-PCR analysis also showed that the laccase mRNA was highly expressed only in the former five strains in the presence of Cu(2+). Taken together, these results indicate that Cu(2+) regulates expression of the laccase gene in a strain-dependent manner. The five strains commonly produced a single predominant laccase protein with a molecular weight of 68 kDa. Peptide sequencing revealed that the laccase was a homolog of Lcc1 of P. coccineus, which was isolated in China. The Cu(2+)-induced culture supernatants exhibited high degradation of polycyclic aromatic hydrocarbons, indicating that the 68-kDa laccase is the primary extracellular degradative enzyme in P. coccineus.

Laccases are multicopper oxidoreductases that can be used in biofuel cells to improve cathode performance by cathodic oxygen reduction. Here we present a laccase from the ligninolytic white-rot fungus Pycnoporus sanguineus that, in contrast to the Trametes versicolor laccase, can be produced in the absence of inducers in a standard culture medium. After 7 days of cultivation the activity of this laccase in culture supernatant reached 2.5 U/ml, which is high enough for direct application of the supernatant in biofuel cells. The highest current density of 115.0 ± 3.5 μA/cm 2 at 400 mV vs. SCE was obtained at pH 5 with a buckypaper cathode with a laccase-containing culture supernatant. The enzyme also showed electrocatalytic activity at pH 6 and 7. These results not only present a new cost-efficient laccase for improving cathode performance, but also show that new laccases with different catalytic properties can be suitable for biofuel cells.

Laccases are enzymes that have the ability to catalyze the oxidation of a wide spectrum of phenolic compounds with the four-electron reduction of molecular oxygen to water. The active site of those proteins contains four copper ions, classified into three types. Laccases are interesting enzymes for study from the point of view of their structure, function and application because of their role in lignin degradation. Structural studies of two thermostable laccases produced by the strain Pycnoporus sanguineus CS43 (PsLacI and PsLacII) were performed. Both isoforms of PsLac show high thermal stability, at 60°C and 50°C, respectively, and they remained active at a high concentration of organic solvents. However, PsLacI has a higher thermal and pH stability and tolerance against inhibitors, and is a more efficient catalyst for ABTS and DMP (laccases substrate) than PsLacII. Based on the determined crystal structures we achieved insights into the structural factors relevant for the enzymatic properties of PsLacI and PsLacII. N-glycosylation site Asn354, which is very often present in structures of fungal laccases from other species, was not present in PsLac. This observation may be of particular significance due to the close distance between Asn354 and the substrate-binding pocket. This results in better access to the hydrophobic cavity for a particular substrate. Furthermore, we identified significant differences in the region of substrate-binding pocket, which confer PsLacI a markedly better performance than PsLacII.

Pycnoporus sanguineus could remove 98.5% ciprofloxacin (CIP), 96.4% norfloxacin (NOR), 100% sulfamethoxazole (SMX), and 100% their mixture through biotransformation within 2 d, while Phanerochaete chrysosporium could only remove 64.5% CIP, 73.2% NOR, and 63.3% SMX through biosorption and biotransformation within 8 d, respectively. The efficiencies of antibiotic bioremoval under co-culture were more than that under the pure culture of P. chrysosporium but less than that under the pure culture of P. sanguineus. However, only 2% CIP and 3% NOR under co-culture were detected in the mycelia. In vitro enzymatic degradation and in vivo cytochrome P450 inhibition experiments revealed that laccase and cytochrome P450 could play roles in the removal of above all antibiotics, while manganese peroxidase could only play role in SMX removal. Transformation products of CIP and NOR under the pure culture of P. chrysosporium could be assigned to three different reaction pathways: (i) defluorination or dehydration, (ii) decarboxylation, and (iii) oxidation of the piperazinyl substituent. Additionally, other pathways, (iv) monohydroxylation, and (v) demethylation or deethylation at position N1 also occurred under the co-culture and pure culture of P. sanguineus. Antibacterial activity of antibiotics could be eliminated after treatments with pure and co-culture of P. chrysosporium and P. sanguineus. The cytotoxicity of the metabolites of SMX and NOR under co-culture was lower than that under the pure culture of P. sanguineus, indicating co-culture is a more environmentally friendly strategy to eliminate SMX and NOR.

International audience; The use of synthetic dyes for laccase induction in vivo has been scarcely explored. We characterized the effect of adding different synthetic dyes to liquid cultures of Pycnoporus sanguineus on laccase production. We found that carminic acid (CA) can induce 722 % and alizarin yellow 317 % more laccase than control does, and they promoted better fungal biomass development in liquid cultures. Aniline blue and crystal violet did not show such positive effect. CA and alizarin yellow were degraded up to 95 % during P. sanguineus culturing (12 days). With this basis, CA was selected as the best inducer and used to evaluate the induction of laccase on solid-state fermentation (SSF), using sugarcane bagasse (SCB) as substrate, in an attempt to reach selective delignification. We found that laccase induction occurred in SSF, and a slight inhibition of cellulase production was observed when CA was added to the substrate; also, a transformation of SCB under SSF was followed by the 13 C cross polarization magic angle spinning (CPMAS) solid-state nuclear magnetic resonance (NMR). Results showed that P. sanguineus can selectively delignify SCB, decreasing aromatic C compounds by 32.67 % in 16 days; O-alkyl C region (polysaccharides) was degraded less than 2 %; delignification values were not correlated with laccase activities. Cellulose-crystallinity index was increased by 27.24 % in absence of CA and 15.94 % when 0.01 mM of CA was added to SCB; this dye also inhibits the production of fungal biomass in SSF (measured as alkyl C gain). We conclude that CA is a good inducer of laccase in liquid media, and that P. sanguineus is a fungus with high potential for biomass delignification.

Production of extracellular laccase by the white-rot fungus Pycnoporus sanguineus was examined in batch submerged cultures in shake flasks, baffled shake flasks and a stirred tank bioreactor. The biomass growth in the various culture systems closely followed a logistic growth model. The production of laccase followed a Luedeking-Piret model. A modified Luedeking-Piret model incorporating logistic growth effectively described the consumption of glucose. Biomass productivity, enzyme productivity and substrate consumption were enhanced in baffled shake flasks relative to the cases for the conventional shake flasks. This was associated with improved oxygen transfer in the presence of the baffles. The best results were obtained in the stirred tank bioreactor. At 28 °C, pH 4.5, an agitation speed of 600 rpm and a dissolved oxygen concentration of ~25 % of air saturation, the laccase productivity in the bioreactor exceeded 19 U L(-1 )days(-1), or 1.5-fold better than the best case for the baffled shake flask. The final concentration of the enzyme was about 325 U L(-1).

The occurrence of sulfamethoxazole (SMX) in aquatic environment is a health concern. The presence of SMX significantly inhibited the laccase activity of Pycnoporus sanguineus with a lower removal efficiency of SMX. Although a laccase system with 2,20-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) eliminated 100% SMX within 6h, ABTS might cause an environmental issue. An alternative to SMX elimination is the coculture of Alcaligenes faecalis and P. sanguineus. The SMX removal efficiency at 48h under the coculture with vitamins was higher than that under their pure culture alone, indicating that a coculture was more efficient in eliminating SMX than a pure culture. Only 1% SMX was detected in mycelia, indicating that SMX elimination is achieved primarily through biotransformation rather than adsorption. Laccase production by the coculture effectively inhibited the accumulations of N4-acetyl-SMX and N-hydroxy-SMX and alleviated the cytotoxicity of SMX transformation products. The mixture of SMX and sulfadiazine inhibited their removal efficiency.

Laccase from Pycnoporus sanguineus CS43 was successfully immobilized onto Immobead-150 and Eupergit-C by covalent binding and by entrapment in LentiKats. The highest immobilization was onto Immobead-150 (97.1±1.2%) compared to the other supports, LentiKats (89±1.1%) and Eupergit-C (83.2±1.4%). All three immobilized enzyme systems showed increased thermostability and better mechanical properties than free laccase. Moreover, after 5 cycles of reuse of these systems, 90% of initial laccase activity was retained. Immobead-150 and LentiKats systems exhibited the highest efficiencies in removal of m-cresol under the combined actions of biodegradation and adsorption, while laccase entrapped in LentiKats showed a high ability for degradation of m-cresol within 24h. In addition, the typical Michaelis-Menten enzymatic model effectively described the kinetic profile of m-cresol degradation by the enzyme entrapped in LentiKats. Based on the results obtained in the present study, it can be established that the immobilized biocatalysts developed here possess significant potential for wastewater treatment.

The uptake of precious metals from electronic waste is of environmental significance and potential commercial value. A facile bioreductive synthesis is described for Au nanoparticles (ca. 20 nm) supported on N-doped carbon (Au@NC), which was derived from Au/Pycnoporus sanguineus cells. The interface and charge transport between Au and N-doped carbon were confirmed by HRTEM and XPS. Au@NC was employed as an electrocatalyst for the hydrogen evolution reaction (HER), exhibiting a small onset potential of -54.1 mV (vs. RHE), a Tafel slope of 76.8 mV dec(-1) , as well as robust stability in acidic medium. Au@NC is a multifunctional electrocatalyst, which demonstrates high catalytic activity in the oxygen reduction reaction (ORR), as evidenced by an onset potential of +0.97 V, excellent tolerance toward methanol, and long-term stability. This work exemplifies dual recovery of precious Au and fabrication of multifunctional electrocatalysts in an environmentally benign and application-oriented manner.

Pycnoporus sanguineus is a white-rot basidiomycete that produces laccase as the only oxidoreductase; enzyme synthesis depends on cultivation variables, and fungal species and strain. Laccases have wide substrate specificity, oxidize a broad range of compounds, and show potential for use in dye decolorization. We evaluated laccase production in a recently isolated strain of P. sanguineus cultivated with sugarcane molasses as the only carbon source, and urea or yeast extract as the nitrogen source [at various nitrogen concentrations (0.4, 1.4, 2.4, 3.4, and 4.4 g/L)], supplemented with copper (0, 150, 200, 250, and 300 µM), with or without agitation. The enzymatic extract produced at laccase peak activity was tested for dye decolorization capability on Remazol Brilliant Blue R, Reactive Black 5, Reactive Red 195, and Reactive Yellow 145. The nitrogen source did not affect enzyme production and the higher nitrogen concentration (3.4 g/L nitrogen as urea) increased enzymatic activity. The addition of up to 300 µM of Cu did not affect laccase production, whereas cultivation with agitation increased the activity peak by 17%. The highest laccase activity was ~50,000 U/L on the ninth day of cultivation. After 24 h, decolorization was 80% for Remazol Brilliant Blue R, 9% for Reactive Yellow 145, 6% for Reactive Red 195, and 2% for Reactive Black 5. The enzymatic extract of P. sanguineus provides a potential alternative to wastewater treatment. A better understanding of the behavior of this fungus under various culture conditions would allow improvement of the enzyme production bioprocess.

Laccase production by Pycnoporus sanguineus RP15 grown in wheat bran and corncob under solid-state fermentation was optimized by response surface methodology using a Central Composite Rotational Design. A laccase (Lacps1) was purified and characterized and the potential of the pure Lacps1 and the crude culture extract for synthetic dye decolorization was evaluated. At optimal conditions (eight days, 26 °C, 18% (w/w) milled corncob, 0.8% (w/w) NH₄Cl and 50 mmol·L(-1) CuSO₄, initial moisture 4.1 mL·g(-1)), the laccase activity reached 138.6 ± 13.2 U·g(-1). Lacps1 was a monomeric glycoprotein (67 kDa, 24% carbohydrate). Optimum pH and temperature for the oxidation of 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonate) (ABTS) were 4.4 and 74.4 °C, respectively. Lacps1 was stable at pH 3.0-8.0, and after two hours at 55-60 °C, presenting high redox potential (0.747 V vs. NHE). ABTS was oxidized with an apparent affinity constant of 147.0 ± 6.4 μmol·L(-1), maximum velocity of 413.4 ± 21.2 U·mg(-1) and catalytic efficiency of 3140.1 ± 149.6 L·mmol(-1)·s(-1). The maximum decolorization percentages of bromophenol blue (BPB), remazol brilliant blue R and reactive blue 4 (RB4), at 25 or 40 °C without redox mediators, reached 90%, 80% and 60%, respectively, using either pure Lacps1 or the crude extract. This is the first study of the decolorization of BPB and RB4 by a P. sanguineus laccase. The data suggested good potential for treatment of industrial dye-containing effluents.

We conducted experiments to culture Pycnoporus sp. SYBC-L3 in a medium comprising an industrial waste (dye-containing textile effluent) and a lignocellulosic waste (Phragmites australis) that achieved laccase production while having the color removed from the wastewater. Our experimental results showed that the fungus grew well in liquid submerged cultivation with the diluted textile effluent as the sole culture medium, but relatively low extracellular laccase activity (1.8 U/mL) was produced. Addition of the lignocellulosic biomass enhanced laccase production and color removal. The highest laccase activity was found to be 6.5 U/mL in the presence of Phragmites australis stem. Under this condition, 70 % color removal occurred in the culture medium. This study provided an alternative novel scheme to remove color in textile wastewater while having an economic value added by producing laccase.

This study presents the biodecolorization potential of basidiomycete fungi Trametes hirsuta, Pycnoporus sp., and Irpex sp. for different reactive dyes viz. Reactive Red 120, Remazol Brilliant Blue R (RBBR), Reactive Orange G, and Reactive Orange 16 under static and shaking conditions. The screening trials revealed that T. hirsuta exhibited maximum potential (83.75 %) for biodecolorization of RBBR dye under static conditions after the fifth day of incubation. However, the rate of biodecolorization of RBBR dye by Pycnoporus sp. was much slow and reached maximum (81.25 %) after 15 days of incubation under shaking conditions. By process optimization, enhanced decolorization (91.2 %) of RBBR by T. hirsuta was achieved at pH 5.5 within 24 h using a defined salt medium amended with p-coumaric acid under static conditions. pH was found to be an important parameter for the enzymatic system involved in RBBR dye decolorization by T. hirsuta and Pycnoporus sp. Biodecolorization of RBBR dye was determined by a reduction in optical density at the wavelength of maximum absorbance (λ, 578 nm) by UV-vis spectrophotometer. The shift in maximum wavelength toward shorter/longer wavelength in UV-vis scanning spectrum revealed the degradation of RBBR dye into different transformation products.

The State of Hidalgo (Mexico) has a large area of forests known as the Huasteca Hidalguense, with a large variety of microorganisms inhabiting it. They represent an important resource from the ecological and technological point of view because they can be used in a broad variety of industrial processes. Due to the climatic conditions of this region, fungi inhabiting it must be thermophile or, at least, thermotolerant, as temperatures can be higher than 45°C in the summer, declining to 20°C in the winter. Use of ligninolytic fungi relies on their capacity to produce enzymes of industrial interest, a topic that has been under continuous research by academic and industrial investigators. Among the most important enzymes are proteases that are widely used due to their biotechnological applications with a high economic impact. Other enzymes, laccases, peroxidases, and lipases are of interest for the industries of the state of Hidalgo, especially in the textile industry, specifically in effluent processing. Fungi (n=156) were collected in the Huasteca Hidalguense, of which 100 were isolated in potato-dextrose-agar covered plates and maintained in tilted tubes. Afterwards, enzymatic activity (laccase, protease and lipase) was determined in the plates. The purpose was to select those fungi with the highest potential for biotechnological applications. Fungi generally grew at either 30°C or 37°C, and for some isolates enzymatic activities were detected at this higher temperature. Results are presented as the relation between enzymatic activity and growth rate: 60 fungi presented laccase activity, 49 had lipase activity, and none had protease activity. In most cases, enzymatic activity was higher than the growth rate, indicating that the isolated fungi have a great biotechnological potential. Statistical analysis revealed that isolates 31 (Trametes) and 8.1 (unidentified) have a larger potential to be studied as laccase-producing fungi. On the other hand, isolates 144.2 (Fomes), 154 (Trametes), and 147.2 (Pycnoporus) are of interest as lipase activity producers, an activity scarcely studied in this type of microorganisms.

Co-cultivation of mutant Penicillium oxalicum SAU(E)-3.510 and Pleurotus ostreatus MTCC 1804 was evaluated for the production of xylanase-laccase mixture under solid-state fermentation (SSF) condition. Growth compatibility between mutant P. oxalicum SAU(E)-3.510 and white rot fungi (P. ostreatus MTCC 1804, Trametes hirsuta MTCC 136 and Pycnoporus sp. MTCC 137) was analyzed by growing them on potato dextrose agar plate. Extracellular enzyme activities were determined spectrophotometrically. Under derived conditions, paired culturing of mutant P. oxalicum SAU(E)-3.510 and P. ostreatus MTCC 1804 resulted in 58% and 33% higher levels of xylanase and laccase production, respectively. A combination of sugarcane bagasse and black gram husk in a ratio of 3:1 was found to be the most ideal solid substrate and support for fungal colonization and enzyme production during co-cultivation. Maximum levels of xylanase (8205.31 ± 168.31 IU g(-1)) and laccase (375.53 ± 34.17 IU g(-1)) during SSF were obtained by using 4 g of solid support with 80% of moisture content. Furthermore, expressions of both xylanase and laccase were characterized during mixed culture by zymogram analysis. Improved levels of xylanase and laccase biosynthesis were achieved by co-culturing the mutant P. oxalicum SAU(E)-3.510 and P. ostreatus MTCC 1804. This may be because of efficient substrate utilization as compared to their respective monocultures in the presence of lignin degradation compounds because of synergistic action of xylanase and laccase. Understanding and developing the process of co-cultivation appears productive for the development of mixed enzyme preparation with tremendous potential for biobleaching.

Textile and Paper Engineering Department. ETSEIAT. Universitat Politècnica de Catalunya. Colom 11. E-08222 Terrassa, Spain Instituto de Recursos Naturales y Agrobiología de Sevilla, CSIC, P.O. Box 1052. E-41080 Seville, Spain Centro de Investigaciones Biológicas, CSIC, Ramiro de Maeztu 9. E-28040 Madrid, Spain, Flax and sisal pulps were treated with two laccases (from Pycnoporus cinnabarinus, PcL and Trametes villosa, TvL respectively), in the presence of different phenolic compounds (syringaldehyde, acetosyringone and p-coumaric acid in the case of flax pulp, and coniferaldehyde, sinapaldehyde, ferulic acid and sinapic acid in the case of sisal pulp). In most cases the enzymatic treatments resulted in increased kappa number of pulps suggesting the incorporation of the phenols into fibres. The covalent binding of these compounds to fibres was evidenced by the analysis of the treated pulps, after acetone extraction, by pyrolysis coupled with gas chromatography/mass spectrometry in the absence and/or in the presence of tetramethylammonium hydroxide (TMAH) as methylating agent. The highest extents of phenol incorporation were observed with the p-hydroxycinnamic acids p-coumaric and ferulic acids. The present work shows for the first time the use of analytical pyrolysis as an effective approach to study fibre functionalisation by laccase-induced grafting of phenols., This work was supported by a Spanish MEC projects TECHFIBER (CTQ2007-68003-C02-01), FUNCICEL (CTQ2009-12904) and ELLE (AGL2008-00709) and a European Integrated Project from Sixth Framework Program BIORENEW (NMP2-CT-2006-026456).

A new white-rot fungus SYBC-L1, which could produce an extracellular laccase, was isolated from a decayed Elaeocarpus sylvestris. The strain was identified as Pycnoporus sp. SYBC-L1 according to the morphological characteristics and ribosomal ITS1-5.8S-ITS2 RNA genomic sequence analysis. The highest laccase activity of 24.1 U ml(-1), which was approximately 40-fold than that in basal medium, was achieved in optimal culture medium in submerged fermentation. The laccase produced by Pycnoporus sp. SYBC-L1 was not only a cold adaptation enzyme with a relative catalytic activity of 30.2% at 0 degrees C but also a high thermostable enzyme. The half-lives at 60, 70 and 80 degrees C were 85.5, 37.2, and 2.6 h, respectively. The laccase could effectively decolorize weak acid blue AS and diamond black PV up to 88% and 74.7%, respectively, within 2 h in the absence of any redox mediators. The results suggested Pycnoporus sp. SYBC-L1 was a potential candidate for laccase production and industrial application.

Laccase from 5 white-rot fungal strains (4 Trametes and 1 Pycnoporus strains) were evaluated in the removal spectra with/without mediators against 11 EDCs. Purified laccase from Trametes sp. was also used to reveal the precise degradation spectra and degradation profiles in time course against 20 EDCs with/without mediators. In addition, effectivity of laccase for the purification of complex EDCs contamination was evaluated combining several EDCs. The removal characteristics among tested strains were almost the same and crude/purified laccase could remove various EDCs. Bis(4-hydroxyphenyl)sulfone, diethylhexylphthalate (DEHP), pyrene (PY), anthracene, 3,5-dichlorophenol and pentachlorophenol could not be removed by laccase. DEHP and PY could not be removed even with mediators. Vanillin and vanillic acid revealed to be possible naturally occurring mediators. Laccase-mediator system could expand the degradation spectrum and enhance the EDCs removal ratio and rate. When complex mixtures of EDCs were treated with laccase, the removal ratio was enhanced in comparison to that of single application. Some coexisting EDCs could act as mediators. Thus, the availability of laccase and the effectivity of a mediator on EDCs treatment were indicated.

Aims: Tyrosinase production by Pycnoporus cinnabarinus and Pycnoporus sanguineus was screened among 20 strains originating from various geographical areas, particularly from tropical environments. The tyrosinase from the most efficient strain was purified and characterized and tested for food additive applications. Methods and Results: Monophenolase and diphenolase activities of tyrosinase were measured from cell lysate from the 20 Pycnoporus strains, for 8–10 days of cultivation. The strain P. sanguineus CBS 614.73 showed the highest productivity (45·4 and 163·6 U g−1 protein per day for monophenolase and diphenolase respectively). P. sanguineus CBS 614.73 tyrosinase was purified from concentrated cell lysate, anion‐exchange, size‐exclusion and hydroxyapatite chromatography, with a final yield of 2% and a purification factor of 35–38. The pure enzyme was a monomere with a molecular mass of 45 kDa and it showed four isoforms or isoenzymes with pI between 4·5–5. No N‐glycosylation was found. The N‐terminal amino acid sequence was IVTGPVGGQTEGAPAPNR. The enzyme was shown to be almost fully active in a pH range of 6–7, in a large temperature range (30–70°C), and was stable below 60°C. The main kinetic constants were determined. The tyrosinase was able to convert p‐tyrosol and p‐coumaric acid into hydroxytyrosol and caffeic acid, respectively, and it could also catalyse the cross‐linking formation of a model protein. Conclusions: Among the genus Pycnoporus, known for the production of laccase, the strain P. sanguineus CBS 614.73 was shown to produce one other phenoloxidase, a new monomeric tyrosinase with a specific activity of 30 and 84 U mg−1 protein for monophenolase and diphenolase respectively. Significance and Impact of the Study: This study identified P. sanguineus CBS 614.73 as a potential producer of a tyrosinase which demonstrated effectiveness in the synthesis of antioxidant molecules and in protein cross‐linking.

The production of thermostable laccases from a native strain of the white-rot fungus Pycnoporus sanguineus isolated in Mexico was enhanced by testing different media and a combination of inducers including copper sulfate (CuSO4). The best conditions obtained from screening experiments in shaken flasks using tomato juice, CuSO4, and soybean oil were integrated in an experimental design. Enhanced levels of tomato juice as the medium, CuSO4 and soybean oil as inducers (36.8% (v/v), 3 mmol/L, and 1% (v/v), respectively) were determined for 10 L stirred tank bioreactor runs. This combination resulted in laccase titer of 143,000 IU/L (2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid), pH 3.0), which represents the highest activity so far reported for P. sanguineus in a 10-L fermentor. Other interesting media resulting from the screening included glucose-bactopeptone which increased laccase activity up to 20,000 IU/L, whereas the inducers Acid Blue 62 and Reactive Blue 19 enhanced enzyme production in this medium 10 times. Based on a partial characterization, the laccases of this strain are especially promising in terms of thermostability (half-life of 6.1 h at 60 °C) and activity titers.

A laccase stage can be used as a pre-treatment of a standard chemical bleaching sequence to reduce environmental concerns associated to this process. The importance of each independent variable and its influence on the properties of the bleached pulp have been studied in depth in this work, using an adaptive network-based fuzzy inference system (ANFIS) with four independent variables (laccase, buffer, mediator and oxygen) as input. Eucalyptus globulus kraft pulp was biobleached using a laccase from Pycnoporus sanguineus and a natural mediator (acetosyringone). Later, an alkaline extraction and a hydrogen peroxide treatment were applied. Most biobleaching processes showed a decrease in kappa number and an increase in brightness with no significant impact on the viscosity values, compared with the control. Oxygen was the variable with the smallest influence on the final pulp properties while the laccase and buffer solution showed a significant influence.

Data on glucose dehydrogenases (GDHs) are scarce and availability of these enzymes for application purposes is limited. This paper describes a new GDH from the fungus Pycnoporus cinnabarinus CIRM BRFM 137 that is the first reported GDH from a white-rot fungus belonging to the Basidiomycota. The enzyme was recombinantly produced in Aspergillus niger, a well-known fungal host producing an array of homologous or heterologous enzymes for industrial applications. The full-length gene that encodes GDH from P. cinnabarinus (PcGDH) consists of 2,425 bp and codes for a deduced protein of 620 amino acids with a calculated molecular mass of 62.5 kDa. The corresponding complementary DNA was cloned and placed under the control of the strong and constitutive glyceraldehyde-3-phosphate dehydrogenase promoter. The signal peptide of the glucoamylase prepro sequence of A. niger was used to target PcGDH secretion into the culture medium, achieving a yield of 640 mg L-1, which is tenfold higher than any other reported value. The recombinant PcGDH was purified twofold to homogeneity in a one-step procedure with a 41 % recovery using a Ni Sepharose column. The identity of the recombinant protein was further confirmed by immunodetection using western blot analysis and N-terminal sequencing. The molecular mass of the native PcGDH was 130 kDa, suggesting a homodimeric form. Optimal pH and temperature were found to be similar (5.5 and 60 A degrees C, respectively) to those determined for the previously characterized GDH, i.e., from Glomerella cingulata. However PcGDH exhibits a lower catalytic efficiency of 67 M-1 s(-1) toward glucose. This substrate is by far the preferred substrate, which constitutes an advantage over other sugar oxidases in the case of blood glucose monitoring. The substrate-binding domain of PcGDH turns out to be conserved as compared to other glucose-methanol-choline (GMCs) oxidoreductases. In addition, the ability of PcGDH to reduce oxidized quinones or radical intermediates was clearly demonstrated, which raises prospects for applying this enzyme to detoxify toxic compounds formed during the degradation of lignin.

The petrol ether, ethyl acetate and methanol extracts of eight medicinal polypore fungi from China were evaluated for cytotoxic activities using MTT-dye assay. All the petrol ether and ethyl acetate extracts exhibited cytotoxicity against human cervix epitheloid carcinoma cell lines (Hela) and human hepatoma cell lines (SMMC-7721). Cytotoxicity activity was also observed in the methanol extracts of Phellinus conchatus and Pycnoporus sanquineus, but the methanol extracts from Cryptoporus volvatus, Fomitopsis pinicola, Fomes hornodermus, Lenzites betulina, Trametes gibbosa and Trametes orientalis showed weak activity when compared with quercetin.

Brazilian tropical fungi were screened for lignin degradation, and the ability to increase or decrease thein vitro digestibility and pH of wheat straw used as a substrate (at 25 and 30 °C after 30 and 60 d of incubation). Out of 72 species and strains ofAgrocybe, Antrodiella, Auricularia, Coriolopsis, Cymatoderma, Fomitopsis, Ganoderma, Gerronema, Gloeophyllum, Gymnopilus, Irpex, Lentinus, Melanoporia, Oligoporus, Oudemansiella, Panaeolus, Peniophora, Phellinus, Pherotus, Psathyrella, Psilocybe, Pycnoporus, Rigidoporus, Schizophyllum, Trametes, Trichaptum andTyromyces, 22 decomposed more than 50% of lignin and 10 increased thein vitro substrate digestibility by more than 30 U. The highest degradation of lignin was observed withLentinus crinitus (80 %, 60 d) and the highest increase inin vitro substrate digestibility was caused byPeniophora utriculosa (36 U, 30 d).

In this study, the thermotolerant yeast Kluyveromyces marxianus CECT 10875 was compared to Saccharomyces cerevisae Ethanol Red for lignocellulosic ethanol production. For it, whole slurry from steam-exploded wheat straw was used as raw material, and two process configurations, simultaneous saccharification and fermentation (SSF) and presaccharification and simultaneous saccharification and fermentation (PSSF), were evaluated. Compared to S. cerevisiae, which was able to produce ethanol in both process configurations, K. marxianus was practically inhibited, and neither growth nor ethanol production occurred during the processes. However, the prior laccase treatment of the whole slurry, which removed specifically the lignin phenols content from the overall inhibitory compounds present in slurry, triggered the fermentation by k. marxianus, attaining ethanol concentrations and yields comparable to those obtained by S. cerevisiae.

This study investigates fungal pretreatment of switchgrass involving solid state fermentation (SSF) to improve saccharification and simultaneously produce enzymes as co-products. The results revealed that the fungus Pycnoporus sp. SYBC-L3 can significantly degrade lignin and enhance enzymatic hydrolysis efficiency. After a 36-d cultivation period, nearly 30% reduction in lignin content was obtained without significant loss of cellulose and hemicellulose, while a considerable amount of laccase, as high as 6.3 U/g, was produced. After pretreatment, pores on switchgrass surface were observed using scanning electron microscopy (SEM). The enzymatic hydrolysis efficiency for the switchgrass with 36-d pretreatment was about 50% greater than the untreated one. Our results suggest that solid state fungal cultivation may be a good method for switchgrass pretreatment, which can simultaneously achieve high efficiency of enzymatic hydrolysis and production of some useful enzymes for other industrial utilization.

The genotoxic potential of solutions of the textile dye “Reactive Black 5” that were subjected to decolorizing treatments with the fungal strains Coriolopsis polyzona MUCL33483 , Penicillium sp. MUBA001 and Pycnoporus sp. MUBA002 was tested. The genotoxicity of the solutions was determined by evaluation of micronuclei formation in Vicia faba root cells and calculation of a damage index (MN ID ). Non-treated Reactive Black 5 solutions (50–1000 ppm) caused a statistically significant increase in micronuclei formation and, by then, in damage index. Solutions of dye treated with C. polyzona MUCL33483 and Pycnoporus sp. MUBA002 showed color loss, probably due to enzymatic breakdown of the colorant, but maintenance or even an increase in genotoxicity. On the other hand, the Penicillium sp. strain MUBA001 caused decolorization of the dye, apparently by adsorption on mycelia, and, for solutions that initially contained 50 ppm of colorant, an elimination of the genotoxicity was observed after three weeks of treatment.

The decolourization of azo dyes by various white rot fungi was studied in the absence of an external carbon source. Although the decolourization abilities of strains used in this study are well documented, these strains have not been studied or compared for their decolourization abilities in the absence of an external carbon source. Decolourization of dyes was also studied in the presence of the external carbon source glucose. Daedalea flavida and Phanerochaete chrysosporium were able to decolourize the dyes in the absence of glucose. D. flavida exhibited a better ability to decolourize the dyes as compared with P. chrysosporium in the absence of glucose. Culture conditions were optimized to enhance the decolourization ability of D. flavida. Shaking of cultures inhibited the decolourization of Amaranth by D. flavida. The optimum culture conditions were 36 degrees C and pH 5.5 for decolourization of Amaranth in a stationary culture condition. We achieved 99% decolourization of Amaranth in 5 days under optimum conditions in the presence of glucose. D. flavida also decolourized the Amaranth with equal efficiency in the absence of glucose. To the best of the authors' knowledge, this is the first paper reporting the decolourization of dyes in the absence of an external carbon source by D. flavida.

The efficiency of the two white-rot fungi Pycnoporus coccineus and Coriolopsis polyzona in the Olive Oil Mill Wastewater (OOMW) treatment was investigated. Both fungi were active in the decolourisation and COD removal of OOMW at 50 g/L COD, but only the first fungus remains effective on the crude effluent (COD=100 g/L). Moreover P. coccineus was less affected by oxygen supplementation and exhibited a high tolerance to agitation in comparison to C. polyzona. However, it required a nitrogen supplementation to obtain faster and higher COD removal. To overcome the negative effect of agitation on fungi growth and efficiency, immobilisation of C. polyzona and P. coccineus in polyurethane foam was applied. The immobilized system showed better COD decreases during three consecutive batches without remarkable loss of performances. The results obtained in this study suggested that immobilized C. polyzona and especially immobilized P. coccineus might be applicable to a large scale for the removal colour and COD of OOMW.

Pycnoporus cinnabarinus laccase and a chimeric laccase-CBM were applied in softwood kraft pulp biobleaching in the presence of 1-hydroxybenzotriazole (HBT). The presence of CBM could enhance the laccase biobleaching potential as a decrease in the enzymatic charge and chlorine dioxide consumption, as well as an increase in pulp brightness were observed. Laccase/HBT treatment could be improved by increasing oxygen pressure from 1 to 3bar and pulp consistency from 5% to 10%. Conversely, under the same conditions, no improvement of laccase-CBM/HBT treatment was observed, indicating a different behavior of both systems. However, laccase-CBM/HBT treatment led to a better preservation of pulp properties. This effect was probably due to fiber surface modifications involving the action of the CBM. Transmission electron microscopy examination of pulp fibers indicated a retention of laccase-CBM inside the pulp fibers due to CBM binding and an increased external microfibrillation of the fibers due to enzymatic treatments.

In this work, kenaf pulp was delignified by using laccase in combination with various redox mediators and the efficiency of the different laccase–mediator systems assessed in terms of the changes in pulp properties after bleaching. The oxidative ability of the individual mediators used (acetosyringone, syringaldehyde, p-coumaric acid, vanillin and actovanillone) and the laccase–mediator systems was determined by monitoring the oxidation–reduction potential (ORP) during process. The results confirmed the production of phenoxy radicals of variable reactivity and stressed the significant role of lignin structure in the enzymatic process. Although changes in ORP were correlated with the oxidative ability of the mediators, pulp properties as determined after the bleaching stage were also influenced by condensation and grafting reactions. As shown here, ORP measurements provide a first estimation of the delignification efficiency of a laccase–mediator system.

International audience; The Pycnoporus fungi are white-rot basidiomycetes listed as food- and cosmetic-grade microorganisms. Three high redox potential laccases from Pycnoporus coccineus and Pycnoporus sanguineus were tested and compared, with the commercial Suberase® as reference, for their ability to synthesise natural active oligomers from rutin (quercetin-3-rutinoside, one of the best-known naturally occurring flavonoid glycosides). The aim of this work was to develop a process with technical parameters (solvent, temperature, reaction time and raw materials) that were easy to scale up for industrial production and compatible with cosmetic and pharmaceutical formulation guidelines. The aqueous mixture of glycerol/ethanol/buffer described in this study met this requirement and allowed the solubilisation of rutin and its oxidative bioconversion into oligomers. The four flavonoid oligomer mixtures synthesised using laccases as catalysts were analysed by high performance liquid chromatography–diode array detection–negative electrospray ionisation–multistage mass spectrometry. Their chromatographic elution profiles were compared and 16 compounds were characterised and identified as dimers and trimers of rutin. The oligorutins were different in Suberase® and Pycnoporus laccase reaction mixtures. They were evaluated for their antioxidant, anti-inflammatory and anti-ageing activities on specific enzymatic targets such as cyclooxygenase (COX-2) and human matrix metalloproteinase 3 (MMP-3). Expressed in terms of IC50, the flavonoid oligomers displayed a 2.5- to 3-fold higher superoxide scavenging activity than monomeric rutin. Pycnoporus laccase and Suberase® oligorutins led to an inhibition of COX-2 of about 35% and 70%, respectively, while monomeric rutin showed a near-negligible inhibition effect, less than about 10%. The best results on MMP-3 activity were obtained with rutin oligomers from P. sanguineus IMB W006-2 laccase and Suberase® with about 70–75% inhibition