A GH115 alpha-glucuronidase from Schizophyllum commune contributes to the synergistic enzymatic deconstruction of softwood glucuronoarabinoxylan

Background: Lignocellulosic biomass from softwood represents a valuable resource for the production of biofuels and bio-based materials as alternatives to traditional pulp and paper products. Hemicelluloses constitute an extremely heterogeneous fraction of the plant cell wall, as their molecular structures involve multiple monosaccharide components, glycosidic linkages, and decoration patterns. The complete enzymatic hydrolysis of wood hemicelluloses into monosaccharides is therefore a complex biochemical process that requires the activities of multiple degradative enzymes with complementary activities tailored to the structural features of a particular substrate. Glucuronoarabinoxylan (GAX) is a major hemicellulose component in softwood, and its structural complexity requires more enzyme specificities to achieve complete hydrolysis compared to glucuronoxylans from hardwood and arabinoxylans from grasses. Results: We report the characterisation of a recombinant α-glucuronidase (Agu115) from Schizophyllum commune capable of removing (4-O-methyl)-glucuronic acid ((Me)GlcA) residues from polymeric and oligomeric xylan. The enzyme is required for the complete deconstruction of spruce glucuronoarabinoxylan (GAX) and acts synergistically with other xylan-degrading enzymes, specifically a xylanase (Xyn10C), an α-l-arabinofuranosidase (AbfA), and a β-xylosidase (XynB). Each enzyme in this mixture showed varying degrees of potentiation by the other activities, likely due to increased physical access to their respective target monosaccharides. The exo-acting Agu115 and AbfA were unable to remove all of their respective target side chain decorations from GAX, but their specific activity was significantly boosted by the addition of the endo-Xyn10C xylanase. We demonstrate that the proposed enzymatic cocktail (Agu115 with AbfA, Xyn10C and XynB) achieved almost complete conversion of GAX to arabinofuranose (Araf), xylopyranose (Xylp), and MeGlcA monosaccharides. Addition of Agu115 to the enzymatic cocktail contributes specifically to 25 % of the conversion. However, traces of residual oligosaccharides resistant to this combination of enzymes were still present after deconstruction, due to steric hindrances to enzyme access to the substrate. Conclusions: Our GH115 α-glucuronidase is capable of finely tailoring the molecular structure of softwood GAX, and contributes to the almost complete saccharification of GAX in synergy with other exo- and endo-xylan-acting enzymes. This has great relevance for the cost-efficient production of biofuels from softwood lignocellulose.Lauren S. McKee, Hampus Sunner, George E. Anasontzis, Guillermo Toriz, Paul Gatenholm, Vincent Bulone, Francisco Vilaplana and Lisbeth Olsso

Signal Processing for Deep-Sea Observatories with Reconfigurable Hardware

The recent evolution of deep-sea observatories has provided the infrastructure for studying rare phenomena in astroparticle physics, extended phenomena in physical oceanography and environmental monitoring for climate modeling and civic alert systems. The observatories involve sets of sensors distributed in the deep-sea, which transmit data through Gbit electro-optical lines to a shore station for real-time processing. Each set of sensors communicates data and control with the shore station through a readout system. Targeting the improvement of the observatory, the current paper proposes a readout system with enhanced functionality, which includes the ability to reconfigure the communication channels, provide statistic measurements of the communicated data and efficient data filtering. The design of the architecture is suited for FPGA implementation and the instantiation on the Xilinx ML605 board validates the results

Metabolic engineering of <em>Fusarium oxysporum</em> to improve its ethanol-producing capability

International audienceFusarium oxysporum is one of the few filamentous fungi capable of fermenting ethanol directly from plant cell wall biomass. It has the enzymatic toolbox necessary to break down biomass to its monosaccharides and, under anaerobic and microaerobic conditions, ferments them to ethanol. Although these traits could enable its use in consolidated processes and thus bypass some of the bottlenecks encountered in ethanol production from lignocellulosic material when Saccharornyces cerevisiae is used namely its inability to degrade lignocellulose and to consume pentoses-two major disadvantages of F. oxysporum compared to the yeast its low growth rate and low ethanol productivity hinder the further development of this process. We had previously identified phosphoglucomutase and transaldolase, two major enzymes of glucose catabolism and the pentose phosphate pathway, as possible bottlenecks in the metabolism of the fungus and we had reported the effect of their constitutive production on the growth characteristics of the fungus. In this study, we investigated the effect of their constitutive production on ethanol productivity under anaerobic conditions. We report an increase in ethanol yield and a concomitant decrease in acetic acid production. Metabolomics analysis revealed that the genetic modifications applied did not simply accelerate the metabolic rate of the microorganism; they also affected the relative concentrations of the various metabolites suggesting an increased channeling toward the chorismate pathway, an activation of the gamma-aminobutyric acid shunt, and an excess in NADPH regeneration

Homologous overexpression of xylanase in Fusarium oxysporum increases ethanol productivity during consolidated bioprocessing (CBP) of lignocellulosics

In an effort to increase ethanol productivity during the consolidated bioprocessing (CBP) of lignocellulosics by Fusarium oxysporum, we attempted the constitutive homologous overexpression of one of the key process enzymes, namely an endo-xylanase. The endo-β-1,4-xylanase 2 gene was incorporated into the F. oxysporum genome under the regulation of the gpdA promoter of Aspergillus nidulans. The transformation was effected through Agrobacterium tumefaciens and resulted in 12 transformants, two of which were selected for further study due to their high extracellular xylanase activities under normally repressing conditions (glucose as sole carbon source). During natural induction conditions (growth on xylan) though, the extracellular enzyme levels of the transformants were only marginally higher (5-10%) compared to the wild type despite the significantly stronger xylanase 2 mRNA signals. SDS-PAGE verified enzyme assay results that there was no intracellular xylanase 2 accumulation in the transformants, suggesting the potential regulation in a post transcriptional or translational level. The fermentative performance of the transformants was evaluated and compared to that of the wild type in simple CBP systems using either corn cob or wheat bran as sole carbon sources. Both transformants produced approximately 60% more ethanol compared to the wild type on corn cob, while for wheat bran this picture was repeated for only one of them. This result is attributed to the high extracellular xylanase activities in the transformants&apos; fermentation broths that were maintained 2-2.5-fold higher compared to the wild type. © 2011 Elsevier B.V

Optical calibration from tens to hundreds of meters for a neutrino telescope

A critical review of the available optical calibration techniques is given in the context of the Neutrino Burst Experiment. We will discuss the effects of the optical properties of water on different candidate light sources and the challenges from an engineering point of view. The merits of candidate light sources will be compared. © 2009 Elsevier B.V. All rights reserved

Lipases efficiently stearate and cutinases acetylate the surface of arabinoxylan films

This is the first report on successful enzyme catalyzed surface esterification of hemicellulose films. Enzyme catalyzed surface acetylation with vinyl acetate and stearation with vinyl stearate were studied on rye arabinoxylan (AX) films. Different surface analytical techniques (FT-IR, TOF-SIMS, ESCA, CA) show that lipases from Mucor javanicus, Rhizopus oryzae and Candida rugosa successfully surface stearate AX films and that a cutinase from Fusarium solani pisi surface acetylates these films. The specificities of cutinase and lipases were also compared, and higher activity was observed for lipases utilizing long alkyl chain substrates while higher activity was observed for cutinase utilizing shorter alkyl chain substrates. The contact angle analysis showed films with increased initial hydrophobicity on the surfaces.Anne Wendel, Anders Martensson and Per Malmberg from Chalmers University of Technology are acknowledged for their help in the ESCA, FT-IR and SIMS measurements. The Knut and Alice Wallenberg Foundation is gratefully acknowledged for donating a base for a Swedish Research Center, the Wallenberg Wood Science Center, thus financing this work