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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.

Fungi are an important source of traditional medicine in Asian and Western countries. Globally, cancer is one of the leading causes of death with 22 million cases predicted by 2030. The cytotoxicity of 13 South African macrofungal species against HeLa, HT-29, MCF-7, MIA PaCa-2 and PC-3 cancer cell lines was measured using the 3-(4,5-dimethyl-2-thiazolul)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay, and the IC 50 values determined for the most cytotoxic macrofungal species. Fomitopsis lilacinogilva had IC 50 values of 42.2, 55.1, 63, 48.8 and 79.8 μg/mL against the HeLa, HT-29, MCF-7, MIA PaCa-2 and PC-3 cancer cell lines, respectively. Gymnopilus junonius had IC 50 values of 55.1 and 52.5 μg/mL against the HeLa and MIA PaCa-2 cancer cell lines, respectively. Pycnoporus sanguineus had IC 50 values of 24.2, 48.1, 32.7, F. lilacinogilva , G. junonius and P. sanguineus was confirmed using Hoechst and phalloidin staining. Morphological changes included condensed chromatin, membrane blebbing, loss of cytoskeletal arrangement and rounding up of cells, as well as multi- and micronuclei. Macrofungal research in South Africa remains a novel field, and this study provides interesting data on the cytotoxicity of South African macrofungal species against some of the most common cancers affecting humans worldwide.

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 .

Emerging contaminants like hormones can interfere ecological life health. Both liquid cultivation and direct enzymatic process have been proved effective for hormonal removal. This study aimed to assess direct removal of 17 β -estradiol (E2) from solid medium poultry litter (PL) by cultivating laccase-producing fungi toward clean treatment of PL. Before E2 removal test, laccase production was optimized by addition of citric acid and lignocellulosic biomasses into PL, which turned out to dramatically enhance laccase production to varying degrees. The highest laccase activity of approximately 16 U/g medium was produced by Pycnoporus sp. SYBC-L3 on PL mixed with sweat sorghum bagasse at a 2 to 3 (g/g) ratio. An extra dose of E2 spiked in the PL/biomass mixture (final concentration of 10 μg/g) was used to test its effect on laccase production and subsequent E2 removal. Over 80% E2 was removed from the above cultivation system, and E2 removal appeared to be positively correlated with laccase activity. Our results might present an alternative clean strategy for livestock manure treatment before land application.

A unique, non-leaching and durable novel anti-biocide polyacrylamide-starch coating (pAam-g-S) has successfully been synthesized. In this study, a grafted cationic-functionalized polymeric coating was designed and cross-linked with native starch extracted from Dioscorea hispida sp. yam to afford multifunctional anti-fungal and anti-termite coating. Leaching of the disinfectant into the environment was eliminated because no toxic chemicals were used. The copolymerization was successfully carried out to produce various ratios of pAam-g-S coating using modification of starch-gel. Results obtained from Fourier Transform Infrared-Attenuated Total Reflectance (FTIR-ATR) confirmed the monomer pAam was grafted onto the starch backbone as shown by the cross-linked peak at 1642 cm−1. Supported by differential scanning calorimetry (DSC), the highest transition glass temperature was observed at 154 °C. The coating was designed to continuously decontaminate against pathogenic fungus and termite in addition to afford preliminary anti-biocides properties. Facile anti-fungal and anti-termite evaluations were conducted via total weight loss of wood (%) studies using white rot fungus, Pycnoporus. sanguineus and termite Coptotermes curvignathus respectively. In order to verify the coating performance, gel fractions, morphologies and heat resistant properties were also investigated.

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.

Laccase has been emerged as a valuable green biocatalyst in cleaner production, which requires vast amount of laccase at low production cost and with good properties. The current work evaluated a wild strain Pycnoporus sp. SYBC-L3 as an efficient microbial cell factory for laccase production. High-cell density cultivation (HCDC) of L3 was developed in a 5-L bioreactor by adjusting the ratio of inoculum to medium, achieving over 4-fold of both laccase activity (240 U/mL) and fungal biomass (20 g/L) than those obtained under routine procedures. Laccase productivity was further assessed at pilot-scale in a 500-L and a 5-ton bioreactor, where the highest activity of around 60 U/mL and 80 U/mL were produced, respectively. Cost percentage analysis for the 5-ton reactor demonstrated that labor was the highest portion (50%) in total cost for industrial production in this case. Using a one-step purification scheme, laccase was purified to homogeneity from the culture broth with yield of 44% and 97% purity. Circular dichroism (CD) analyses suggested that the secondary structure of laccase was mainly composed of β-sheet that played a vital role in maintaining laccase activity, and also revealed the potential mechanisms for the changes in laccase activities under varying conditions. Powdered laccase was efficiently prepared from cell-free culture broth using spray drying technique with above 74% activity retained. This study provides important information for an efficient and scalable production of laccase.

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.

A native laccase (Lac-Q) with robust cold-adapted and thermostable characteristics from the white-rot fungus Pycnoporus sp. SYBC-L10 was purified, characterized, and used in antibiotic treatments. Degradation experiments revealed that Lac-Q at 10.0 U mL−1 coupled with 1.0 mmol L−1 ABTS could degrade 100% of the tetracycline or oxytetracycline (50 mg L−1) within 5 min with a static incubation at 0 °C (pH 6.0). The presence of the Mn2+ ion inhibited the removal rate of tetracycline and oxytetracycline by the Lac-Q–ABTS system, and the presence of Al3+, Cu2+, and Fe3+ accelerated the removal rate of tetracycline and oxytetracycline by the Lac-Q–ABTS system. Furthermore, seven transformation products of oxytetracycline (namely TP 445, TP 431, TP 413, TP 399, TP 381, TP 367, and TP 351) were identified during the Lac-Q-mediated oxidation process by using UPLC–MS/MS. A possible degradation pathway including deamination, demethylation, and dehydration was proposed. Furthermore, the growth inhibition of Bacillus altitudinis SYBC hb4 and E. coli by tetracycline antibiotics revealed that the antimicrobial activity was significantly reduced after treatment with the Lac-Q–ABTS system. Finally, seven transformation products of oxytetracycline (namely TP 445, TP 431, TP 413, TP 399, TP 381, TP 367, and TP 351) were identified during the Lac-Q-mediated oxidation process by using UPLC–MS/MS. A possible degradation pathway including deamination, demethylation, and dehydration was proposed. These results suggest that the Lac-Q–ABTS system shows a great potential for the treatment of antibiotic wastewater containing different metal ions at various temperatures.

In this study, we investigated the resistance of C. oleifera Abel. fruit shell's probe to fungi and termites. The degree of decay of C. oleifera Abel. fruit shells inoculated with Coriolus versicolor , Pycnoporus sanguineus , Coniophora puteana and Antrodia spp . in temperate or tropical environment was assessed. The resistance of the shells to subterranean termites in a tropical chamber was also assessed. It was found that there is an overall difference between the resistance to fungi decay and to termite attack. It was found that: the degree of degradation of the shell colonized by white-rot fungi is more severe than the fragmentation of the shell by brown-rot fungi. It was also found that the white-rot decay of C. oleifera Abel. fruit shell is greater than to beech wood, and then brown-rot decay of Scots pine sapwood is severer than to C. oleifera Abel. fruit shell. The resistance of C. oleifera Abel. fruit shell to attack by the subterranean termites was found to be high. It was concluded that the fungal species was an important controlling factor to the decomposition of C. oleifera Abel. fruit shell; and that termite infestation of C. oleifera Abel. fruit shell did take place but termite mortality (100%) occurred. Based on these results, further investigations are being made in order to solve the use of massive by-product from edible tea-seed oil industries.

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.

Numerous fruiting bodies of Mycena species were collected from reed-thatched houses in Northern Germany. The fungi were isolated via spore sowing, purified and then characterized in regard to their ligninolytic enzyme pattern. All fungal isolates were able to decolorize the anthraquinone dye Remazol Brilliant Blue R (RBBR) confirming a ligninolytic enzyme activity. In addition, various Mycena species degraded carboxymethyl cellulose and birch wood xylan to varying extents. The strains produced laccase and in some cases manganese peroxidase, which are considered to be key enzymes involved in lignin degradation. The addition of comminuted reed resulted partly in a considerable increase of the laccase activity in liquid cultures. In extensive long-term experiments on solid media with reed as substrate the Mycena species Mycena arcangeliana, Mycena epipterygia variation epipterygia, Mycena galopus variation leucogala and Mycena polygramma caused a significant weight loss of reed between 7.8 and 17.2 % within 70 d. Because of this, we believe that these Mycena species can be classified as reed-decomposing fungi that may affect the quality of reed roofs.

Cellobiose dehydrogenase (CDH), an extracellular flavocytochrome produced by several wood-degrading fungi, was detected in the culture supernatant of the selective delignifier Pycnoporus sanguineus maintained on a cellulose-based liquid medium. Cellobiose dehydrogenase was purified as two active fractions: CDH1-FAD (flavin domain) (40.4 fold) with recovery of 10.9% and CDH1 (flavo-heme enzyme) (54.7 fold) with recovery of 9.8%. As determined by SDS-PAGE, the molecular mass of the purified enzyme was found to be 113.4kDa and its isoelectric point was 4.2, whereas these values for the FAD-domain were 82.7kDa and pI=6.7. The carbohydrate content of the purified enzymes was 9.2%. In this work, the cellobiose dehydrogenase gene cdh1 and its corresponding cDNA from fungus P. sanguineus were isolated, cloned, and characterized. The 2310bp full-length cDNA of cdh1 encoded a mature CDH protein containing 769 amino acids, which was preceded by a signal peptide of 19 amino acids. Moreover, both active fractions were characterized in terms of kinetics, temperature and pH optima, and antioxidant properties.

This study examined the potential of untreated and alkali-pretreated sugarcane bagasse (SCB) in cellulase, reducing sugar (RS) and fungal biomass production via solid state fermentation (SSF) using Pycnoporus sanguineus. The impact of the composition, structure and cellulase adsorption ability of SCB on the production of cellulase, RS and fungal biomass was investigated. From the morphological and compositional analyses, untreated SCB has relatively more structural changes with a higher percentage of depolymerisation on the cellulose, hemicellulose and lignin content compared to alkali-pretreated SCB. Thus, untreated SCB favoured the production of cellulase and fungal biomass whereas alkali-pretreated SCB yielded a higher amount of RS. The composition and morphology of untreated SCB did not encourage RS production and this suggested that RS produced during SSF might be consumed in a faster rate by the more abundantly grown fungus. Besides that, alkali-pretreated SCB with higher cellulase adsorption ability could have adsorbed the cellulase produced and resulted in a lower cellulase titre. In short, the production of specific bioproducts via SSF is dependent on the structure and composition of the substrate applied.

Laccase is one of the most useful green enzymes for cleaner industrial application and environmental protection. It is necessary to produce laccase with known composition of its commercial style at low production cost in an efficient way. In this study, the efficiency of laccase production by Pycnoporus sp. SYBC-L3 in a 65-L air-lift reactor was evaluated and the main extracellular proteins were identified. The highest laccase activity approximated 72,000 U/L after 6 days' cultivation. Using nano-LC–MS/MS protein identification technology, 22 different peptide fragments were identified matching sequences in different proteins with laccase as the predominant extracellular one. The crude laccase exhibited high catalytic activity at temperatures ranging from 0 °C to 100 °C at an acidic condition, with respective relative activity of 40% at 0 °C and 80% at 100 °C. The crude laccase was found to be very thermal stable, with half-lives of 4 h, 6.5 h and 10 h at 70 °C, 60 °C and 50 °C, respectively. The crude laccase still retained approximately 85% of its initial activity after one year of storage at room temperature. These results showed that laccase can be effectively produced by Pycnoporus sp. SYBC-L3 in air-lift reactor at larger scale and has great potential for industrial production and applications.

Five species of white rot fungi were screened for their capability to synthesize Ag nanoparticles (AgNPs). Three modes of AgNP bioreduction were developed. Pycnoporus sanguineus is found as a potential candidate for the synthesis of AgNPs with a yield at 98.9%. The synthesized AgNPs were characterized using UV–vis spectroscopy, DLS, FTIR, TEM, and SEM. Results showed that AgNP absorption band was located at a peak of 420 nm. Both the SEM and TEM confirmed that the formation of AgNPs were mainly spherical with average diameters of 52.8–103.3 nm. The signals of silver atoms' presence in the mycelium were observed by SEM-EDS spectrum.

International audience; Extractives that provide natural resistance to Bagassa guianensis Aubl. heartwood were examined. Soil-bed tests showed that the B. guianensis heartwood resistance was significantly reduced after ethyl acetate extraction, whereas methanol and especially water extractions improved the resistance. The ethyl acetate extract was submitted to a bioguided fractionation, and fractions were tested against one wood-destroying fungal strain (Pycnoporus sanguineus) and two human pathogenic fungal strains (Candida glabrata (yeast) and Trichophyton rubrum (filamentous dermatophyte)). Fraction F7, which exhibited the strongest antifungal activity, was subsequently fractionated by high performance liquid chromatography (HPLC). Six previously described compounds were isolated. Although the two moracins, 6-O-methyl-moracin N (3) and moracin N (4) were the most active against fungal strains with MIC values between 4 and 16 μg ml−1, the isolated compounds showed less or equivalent antifungal activity than the initial fraction. Possible synergism between compounds 3 and 4 and other secondary metabolites have been hypothesized. Our study demonstrated that this extract as a whole might be used as a wood preservation or antimycotic product.

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).

The white-rot fungus Pycnoporus sp. SYBC-L1 produced large amount of laccase in submerged fermentation. Two laccase isozymes (LacI and LacII) were purified using (NH 4 ) 2 SO 4 fractionation, DEAE-cellulose and Sephadex G-100 column chromatography. The molecular masses of LacI and Lac II were 55.89 and 63.07 kDa, respectively by SDS-PAGE. Both the laccases showed acidic pH optima and high catalytic activities at low temperature for oxidations of 2,6-dimethoxyphenol (DMP), 2,2-azino-bis-(3-ethylbenzothiazoline-6-sulfonate acid) (ABTS), syringaldazine and guaiacol. LacI and LacII were not only with high cold adaptation, but also fairly stable at high temperature. The half-lives of LacI at 50, 60 and 70 °C were 69.31, 2.58 and 0.13 h, respectively, whereas LacII was more stable with half-lives of 256.72, 21.00 and 2.06 h respectively. The best substrates for the enzymes were both found to be ABTS, in which the K m values of LacI and LacII were 0.0166 and 0.0435 mM and the catalytic efficiencies were 19640.36 and 31172.64 S −1 mM −1 , respectively. EDTA and low concentration of Cu 2+ and Mn 2+ almost had non-inhibitions on their activities. LacII with syringaldehyde efficiently decolorized Remazol Brilliant Blue R. The high thermostabilities as well as cold adapted properties made Pycnoporus sp. SYBC-L1 laccases to be excellent candidates in harsh industry.

The present work describes the purification and characterization of a novel extracellular polygalacturonase, PGase I, produced by Pycnoporus sanguineus when grown on citrus fruit pectin. This substrate gave enhanced enzyme production as compared to sucrose and lactose. PGase I is an exocellular enzyme releasing galacturonic acid as its principal hydrolysis product as determined by TLC and orcinol-sulphuric acid staining. Its capacity to hydrolyze digalacturonate identified PGase I as an exo -polygalacturonase. SDS-PAGE showed that PGase I is an N -glycosidated monomer. The enzyme has a molecular mass of 42 kDa, optimum pH 4.8 and stability between pH 3.8 and 8.0. A temperature optimum was observed at 50–60 °C, with some enzyme activity retained up to 80 °C. Its activation energy was 5.352 cal mol −1 . PGase I showed a higher affinity towards PGA than citric pectin (Km = 0.55 ± 0.02 and 0.72 ± 0.02 mg ml −1 , respectively). Consequently, PGase I is an exo -PGase, EC 3.2.1.82.

Leptosphaeria maculans, a pathogen of Brassica napus, is unable to invade most wild-type accessions of Arabidopsis thaliana, although several mutants are susceptible. The infection pathway of L. maculans via a non-invasive inoculation method on A. thaliana lms1 (undefined), pmr4-1 (defective in callose deposition), and pen1-1 and pen2-1 (defective in non-host responses to several pathogens) mutants is described. On wild types Col-0 and Ler-0, hyphae are generally arrested at stomatal apertures. A T-DNA insertional mutant of L. maculans (A22) that penetrates stomatal apertures of Col-0 and Ler-0 five to seven times more often than the wild-type isolate is described. The higher penetration frequency of isolate A22 is associated with an increased hypersensitive response, which includes callose deposition. Complementation analysis showed that the phenotype of this isolate is due to T-DNA insertion in an intronless gene denoted as ipa (increased penetration on Arabidopsis). This gene is predicted to encode a protein of 702 amino acids with best matches to hypothetical proteins in other filamentous ascomycetes. The ipa gene is expressed in the wild-type isolate at low levels in culture and during infection of A. thaliana and B. napus.

Pycnoporous sanguineus was identified as a laccase producer when grown on diluted molasses. The single laccase was purified with a purification factor of 967 by ammonium sulphate precipitation, ion exchange and dye affinity chromatography, to a specific activity of 32.9 U/mg. The molecular mass of 58,000 Da, the optimum pH range between 3 and 5 with ABTS, DMP and guaiacol as substrates and the isoelectric point of 6.7, was similar to some other laccases of filamentous fungi. The optimum temperature was 55 °C and the enzyme displayed enhanced thermal stability with a half-life of 170 min at 75 °C. In terms of k cat / K m values for ABTS, syringaldazine and DMP, DMP was the best substrate. Kinetic analysis of fifteen more compounds revealed that, the enzyme was a true laccase with a requirement for a free –OH group with an adjacent free or derivatised –OH. The o -diphenols were preferred above their p- counterparts. Compounds with three adjacent –OH groups displayed higher binding affinities but phloroglucinol, an m -substituted phenol was unreactive. The apparent higher stability of this laccase makes it a good candidate for further investigation into it possible application in biotechnology.

Laccases isolated from Rhus vernicifera Stokes (tree) and Pycnoporus coccineus (fungus) catalyzed the oxidation of isoeugenol ( 1 ) and coniferyl alcohol ( 5 ) in acetone–water (1:1, v/v). These oxidations follow a first order rate law. In general, the rates of Pycnoporus laccase-catalyzed oxidation of 1 and 5 are approximately three and seven times faster than the corresponding rates of Rhus laccase-catalyzed oxidation, respectively. Thus, synthesis for 2-(4-hydroxyphenyl)coumaran type compounds, such as dehydrodiconiferyl alcohol, can be accomplished by Rhus laccase-catalyzed dehydrogenative polymerization of the corresponding 1-(4-hydroxyphenyl)–1-propene derivatives. The reaction proceeds under very mild reaction conditions. The resulting reaction mixtures are chromatographed on a silica gel column to isolate the products in approximately 30–40% yield.

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.

A crystalline preparation of the apoenzyme of isophenoxazine synthase (2-aminophenol: O2 oxidoreductase) has been obtained from Pycnoporus coccincus. The holoenzyme was reconstituted by addition of riboflavin 5′-phosphate and Mn2+. Flavin-adenine dinucleotide did not serve as a cofactor. Conversion of o-aminophenol to 2-amino-3H-isophenoxazin-3-one was stoichiometric and in the absence of Mn2+ required approx. 1 mole of riboflavin 5′-phosphate per mole of o-aminophenol oxidized. In the presence of Mn2+ riboflavin 5′-phosphate acted as a catalyst. Incubation of the enzyme with o-aminophenol generated thiol groups and also made the reaction susceptible to inhibition by sulfhydryl reagents. A variety of reducing substances repressed the activity of the enzyme; inhibition by ascorbic acid could be reversed by increasing the concentration of riboflavin 5′-phosphate and Mn2+. The reaction was also inhibited by substrate at concentrations above 0.6 mM.

A TLC procedure was developed for the separation of phenoxazinone-type pigments. By employing silica gel G as the adsorbent and a solvent system of benzene-ethyl acetate-glacial acetic acid-formic acid (12:6:1:1), the separation of the following reference compounds was accomplished: cinnabarinic acid (Rf 0.35), cinnabarin (0.47), 2-amino-3-oxo-3H-phenoxazine (0.57), tramesanguin (0.67), and phenoxazine (0.99). Examination of the individual acetone extracts of Pycnoporus cinnabarinus (Jacq. ex Fr.) Karst., P. coccineus (Fr.) Bond. and Sing., and P. sanguineus (L. ex Fr.) Murr. by this technique revealed that all three species contained the same pigments. In addition to cinnabarinic acid, cinnabarin, and tramesanguin, two additional phenoxazinone-type pigments (Rf 0.53 and 0.90) were found in the respective carpophores.