A survey of genes encoding H2O2-producing GMC oxidoreductases in 10 Polyporales genomes

15 p.-4 fig.-1 tab.The genomes of three representative Polyporales (Bjerkandera adusta, Phlebia brevispora and a member of the Ganoderma lucidum complex) recently were sequenced to expand our knowledge on the diversity and distribution of genes involved in degradation of plant polymers in this Basidiomycota order, which includes most wood-rotting fungi. Oxidases, including members of the glucose-methanol-choline (GMC) oxidoreductase superfamily, play a central role in the above degradative process because they generate extracellular H2O2 acting as the ultimate oxidizer in both white-rot and brown-rot decay. The survey was completed by analyzing the GMC genes in the available genomes of seven more species to cover the four Polyporales clades. First, an in silico search for sequences encoding members of the aryl-alcohol oxidase, glucose oxidase, methanol oxidase, pyranose oxidase, cellobiose dehydrogenase and pyranose dehydrogenase families was performed. The curated sequences were subjected to an analysis of their evolutionary relationships,followed by estimation of gene duplication/ reduction history during fungal evolution. Second,the molecular structures of the near one hundred GMC oxidoreductases identified were modeled to gain insight into their structural variation and expected catalytic properties. In contrast to ligninolytic peroxidases,whose genes are present in all white-rot Polyporales genomes and absent from those of brown-rot species, the H2O generating oxidases are widely distributed in both fungal types. This indicates that the GMC oxidases provide H2O2 for both ligninolytic peroxidase activity (in white-rot decay) and Fenton attack on cellulose (in brown-rot decay), after the transition between both decay patterns in Polyporales occurred.This work was supported by the INDOX (www.indoxproject.eu; KBBE-2013-7-613549) European project and by the Spanish HIPOP (BIO2011-26694) project. The work conducted by the U.S. Department of Energy Joint Genome Institute was supported by the Office of Science of the U.S. Department of Energy under Contract DE-AC02-05CH11231, in the frame of the JGI Saprotrophic Agaricomycotina project coordinated by D.S. Hibbett (Clark University, USA).Peer reviewe

Melanins and lipids in Lycoperdon perlatum fruit bodies

5 pages, 1 figure, 3 tables, 27 references.After sequential degradation with sodium persulphate and potassium permanganate, the characteristics of the dark pigments from the soil gasteromycete Lycoperdonperlatum were described. Several aromatic acids (phenolic and benzenecarboxylic) were detected by glc-ms of the degradation products, but the aliphatic content of these polymer fractions was predominant, containing a high proportion of protein and showing fatty acids similar to those previously found in the lipid fraction. An acid soluble fraction of melanin, of a highly aliphatic nature, was also present in Lycoperdon fruit bodies.Peer reviewe

Experimental recreation of the evolution of lignin-degrading enzymes from the Jurassic to date

[Background] Floudas et al. (Science 336: 1715) established that lignin-degrading fungi appeared at the end of Carboniferous period associated with the production of the first ligninolytic peroxidases. Here, the subsequent evolution of these enzymes in Polyporales, where most wood-rotting fungi are included, is experimentally recreated using genomic information.[Results] With this purpose, we analyzed the evolutionary pathway leading to the most efficient lignin-degrading peroxidases characterizing Polyporales species. After sequence reconstruction from 113 genes of ten sequenced genomes, the main enzyme intermediates were resurrected and characterized. Biochemical changes were analyzed together with predicted sequences and structures, to understand how these enzymes acquired the ability to degrade lignin and how this ability changed with time. The most probable first peroxidase in Polyporales would be a manganese peroxidase (Mn3+ oxidizing phenolic lignin) that did not change substantially until the appearance of an exposed tryptophan (oxidizing nonphenolic lignin) originating an ancestral versatile peroxidase. Later, a quick evolution, with loss of the Mn2+-binding site, generated the first lignin peroxidase that evolved to the extant form by improving the catalytic efficiency. Increased stability at acidic pH, which strongly increases the oxidizing power of these enzymes, was observed paralleling the appearance of the exposed catalytic tryptophan.[Conclusions] We show how the change in peroxidase catalytic activities meant an evolutionary exploration for more efficient ways of lignin degradation by fungi, a key step for carbon recycling in land ecosystems. The study provides ancestral enzymes with a potential biotechnological interest for the sustainable production of fuels and chemicals in a biomass-based economy.We acknowledge support of the publication fee by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI), and the EC OpenAIRE FP7 post-grant Open Access Pilot.This work was supported by the INDOX (KBBE-2013-613549) and EnzOx2 (H2020-BBI-PPP-2015-2-720297) EU projects and the NOESIS (BIO2014-56388-R) project of the Spanish Ministry of Economy and Competitiveness (MINECO). The work conducted by JGI was supported by the Office of Science of the U.S. Department of Energy under Contract DE-AC02-05CH11231.EUR 1,620 APC fee funded by the EC FP7 Post-Grant Open Access Pilo

Enzymatic deinking of secondary fibers: cellulases/hemicellulases versus laccase-mediator system

The use of enzymes has been suggested as an environmentally-friendly alternative to complement conventional chemical deinking in the recycling of recovered paper. This study compares the use of carbohydrate hydrolases versus laccase-mediator system for deinking printed fibers from newspapers and magazines. For this purpose, two commercial enzyme preparations with endoglucanase and endoxylanase activities (Viscozyme Wheat from Aspergillus oryzae and Ultraflo L from Humicola insolens) and a commercial laccase NS51002 from Trametes villosa), the latter in the presence of synthetic or natural (lignin related) mediators, were evaluated. The enzymatic treatments were studied at laboratory scale, using a standard chemical deinking sequence consisting of pulping, alkaline deinking and peroxide bleaching stages. Then, handsheets were prepared and their brightness, residual ink concentration, and strength properties were measured. Among the different enzymatic treatments assayed, both carbohydrate hydrolases were found to deink the secondary fibers more efficiently. Brightness increased up to 3-4% ISO on newspaper fibers, being Ultraflo 20% more efficient in the ink removal. Up to 2.5% ISO brightness increase was obtained when magazine fibers were used, being Viscozyme 9% more efficient in the ink removal. As regards laccase-mediator system, alone or combined with carbohydrate hydrolases, it was ineffective deinking both newspaper and magazine fibers, resulting in pulps with worse brightness and residual ink concentration values. However, pulp deinking by laccase-mediator system was displayed when secondary fibers rich in lignin, i.e. printed cardboard, were used, obtaining up to 3% ISO brightness increase and lower residual ink concentrations

Production of new unsaturated lipids during wood decay by ligninolytic basidiomycetes

Instituto de Recursos Naturales y Agrobiología, Consejo Superior de Investigaciones Científicas, E-41080, Seville,1 and Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, E-28006 Madrid,2 SpainLipids were analyzed by gas chromatography-mass spectrometry for a 7-week in vitro decay of eucalypt wood by four ligninolytic basidiomycetes. The sound wood contained up to 75 mg of lipophilic compounds per 100 g of wood. Hydrolysis of sterol esters, which represented 38% of total wood lipids, occurred during the fungal decay. The initial increase of linoleic and other free unsaturated fatty acids paralleled the decrease of sterol esters. Moreover, new lipid compounds were found at advanced stages of wood decay that were identified from their mass spectra as unsaturated dicarboxylic acids consisting of a long aliphatic chain attached to the C-3 position of itaconic acid. These dicarboxylic acids were especially abundant in the wood treated with Ceriporiopsis subvermispora (up to 24 mg per 100 g of wood) but also were produced by Phlebia radiata, Pleurotus pulmonarius, and Bjerkandera adusta. We hypothesize that three main alkylitaconic acids (tetradecylitaconic, cis-7-hexadecenylitaconic, and hexadecylitaconic acids) are synthesized by fungi in condensation reactions involving palmitic, oleic, and stearic acids. We suggest that both wood unsaturated fatty acids (present in free form or released from esters during natural decay) and unsaturated metabolites synthesized by fungi could serve as a source for peroxidizable lipids in lignin degradation by white rot basidiomycetes.This study was supported by the European Commission (contract QLK5-99-1357) and the Spanish CICYT (FEDER project 1FD97-0742).Peer reviewe

Immobilization of Pycnoporus coccineus laccase on Eupergit C: stabilization and treatment of olive oil mill wastewaters

The use of olive oil mill wastewaters (OMW) as organic fertilizer is limited by its phytotoxic effect, due to the high concentration of phenolic compounds. As an alternative to physico-chemical methods for OMW detoxification, the laccase from Pycnoporus coccineus, a white-rot fungus that is able to decrease the chemical oxygen demand (COD) and colour of the industrial effluent, is being studied. In this work, the P. coccineus laccase was immobilized on two acrylic epoxy-activated resins, Eupergit C and Eupergit C 250L. The highest activity was obtained with the macroporous Eupergit C 250L, reaching 110 U g-1 biocatalyst. A substantial stabilization effect against pH and temperature was obtained upon immobilization. The soluble enzyme maintained ≥80% of its initial activity after 24 h at pH 7.0-10.0, whereas the immobilized laccase kept the activity in the pH range 3.0-10.0. The free enzyme was quickly inactivated at temperatures above 50 oC, whereas the immobilized enzyme was very stable up to 70 oC. Gel filtration profiles of the OMW treated with the immobilized enzyme (for 8 h at room temperature) showed both degradation and polymerization of the phenolic compounds.We thank Dr. J. Martinez and T. de la Rubia (University of Granada, Spain) for giving us lyophilized OMW. We thank Thomas Boller (Degussa, Darmstadt, Germany) for supplying Eupergit C samples and for technical help. The authors thank the financial support received from the Spanish Projects BIO2003-00621, VEM2004-08559 and CAM S-0505/AMB0100.Peer reviewe

Oxidative modification of paper pulp lipophilic extractives by laccase-mediator system

Recently, we have shown the effectiveness of the laccase-mediator system in removing lipophilic extractives from pulp regardless the pulping process and the raw material used. This paper summarizes our research findings on the chemistry of the reactions of the main lipophilic extractives present in paper pulps with the laccase-mediator system. The results attained in reactions of several model lipids - including alkanes, fatty alcohols, fatty acids, resin acids, free sterols, sterol esters and triglycerides - with a fungal laccase in the presence of HBT as mediator, are discussed in the context of enzymatic control of pitch deposits, to explain the removal of complex lipid mixtures during laccase-mediator treatment of different pulp types, including eucalypt pulp.This study was funded by the BIORENEW EU-project (NMP2-CT-2006-026456) and the Spanish MICINN (AGL-2008-00709). Beldem (Andenne, Belgium) is acknowledged for laccase supply. S. Molina and J. Rencoret are acknowledged for their contribution to this work.Peer reviewe

Enhanced degradation of softwood versus hardwood by the white-rot fungus Pycnoporus coccineus

16 p.-8 fig.-1 tab. Courier, Marie et al.This study was also funded by the French National Research Agency (A*MIDEX project ANR11-IDEX-0001-02;ANR FUNLOCK ANR-13-BIME-0002-01). The work by the US Department of Energy Joint Genome Institute, a DOE Office of Science User Facility, is supported by the Office of Science of the US Department of Energy under Contract No. DE-AC02-05CH11231.Peer reviewe

Optimization of a laccase-mediator stage for TCF bleaching of flax pulp

7 p.-3 fig.-3 tab.Flax pulp obtained by anthraquinone-soda pulping, resulting in a kappa number of 11.1, a viscosity of 950 ml/g and 36.7% ISO brightness, was bleached in a totally chlorine-free sequence using the enzyme laccase from the fungus Pycnoporus cinnabarinus and 1-hydroxybenzotriazole (HBT) as redox mediator (stage L), followed by a hydrogen peroxide stage (P). The laccase treatment was optimized using a three-variable sequential statistical plan over the following ranges: 1–20 U/g o.d.p. (oven-dried pulp) laccase dose, 0.5–7.5% o.d.p. HBT dose and 1–24 h reaction time. The influence of these variables on several pulp properties after the P stage of the LP sequence was examined.The models defined from the results obtained predicted variations in ISO brightness, viscosity and kappa number of 57.6–74.8%, 590–955 ml/g and 0–6.2, respectively. The variables most strongly influencing these pulp properties were found to be the reaction time and the enzyme dose. A compromise was adopted as regards the operating conditions in order to ensure optimum results. The study was completed by conducting a biobleaching assay in a pressurized reactor (590 kPa) to assess the effect of oxygen pressure. The high pressure level resulted in improved pulp properties by the laccase-mediator system.This work was supported by Spanish CICyT (Project PPQ2000–1068-C02-02, and FEDER Project 2FD97-0896-C02) and Comunidad Autónoma de Madrid, the EU Commission (QLK3-99-590) and the Spanish grant 2002FI 00556.Peer reviewe

Catalytic reforming of dimethyl ether in microchannels

© . This manuscript version is made available under the CC-BY-NC-ND 4.0 license http://creativecommons.org/licenses/by-nc-nd/4.0/The steam reforming and oxidative steam reforming of dimethyl ether (DME) were tested at 573–773¿K over a CuZn/ZrO2 catalyst in microreactors with three different types of channels: ceramic square channels with side lengths of 900 and 400¿µm, and silicon microchannels of 2¿µm of diameter. The channels were first coated with ZrOCl2 (ceramic channels) or Zr(i-PrO)4 (silicon microchannels) and calcined at 773¿K for 2¿h to obtain a homogeneous and well-adhered ZrO2 layer, as determined by SEM, and then Cu and Zn (Cu:Zn¿=¿1:1¿M, 20¿wt% total metal) were co-impregnated. Operation at highly reduced residence time (10-3¿s) while achieving hydrogen yields similar to those recorded over the ceramic channels was possible for the silicon microchannels due to the three orders of magnitude increased contact area. In addition, the amount of catalyst used for coating the silicon microchannels was two orders of magnitude lower with respect to the conventional ceramic channels. Outstanding specific hydrogen production rates of 0.9 LN of H2 per min and cm3 of reactor volume were achieved as well as stable operation for 80¿h, which demonstrates the feasibility of using on-site, on-demand hydrogen generation from DME for portable fuel cell applications.Peer ReviewedPostprint (published version