Compréhension des mécanismes enzymatiques de transformation de la ligine par les champignons filamenteux pour l'obtention des fibres papetières à haute valeur ajoutée

La délignification est un phénomène complexe nécessitant l'interaction de nombreuses enzymes aux capacités catalytiques complémentaires. Les champignons de la pourriture blanche sont les seuls micro-organismes capables de minéraliser la lignine. Pour cela ils sécrètent de nombreuses oxydoréductases lignolytiques comme les laccases et les lignines, manganèse, et versatiles peroxydases ainsi que des enzymes auxiliaires à la délignification comme les xylanases, les féruloyls estérases, les cellobiose déshydrogénases ou les aryl-alcools oxydases. Au cours de cette thèse, nous avons étudié les mécanismes de biotransformation de la lignine par les champignons filamenteux de la pourriture blanche. Pour cela, nous avons choisi comme modèle d'étude le basidiomycète "Pycnoporus cinnabarinus" en raison de sa forte aptitude à dégrader la lignine en ne sécrétant qu'une enzyme lignolytique, la laccase. Nous avons d'une part caractérisé les principales enzymes exocellulaires de "P. cinnabarinus" cultivé en présence de substrats naturels. D'autre part nous nous sommes intéressés plus spécifiquement à la caractérisation de la laccase de ce champignon produite sous forme native ou exprimée par deux hôtes recombinants, "Aspergillus niger" et "Aspergillus oryzae". Après avoir démontré la capacité d'"A. niger" à produire de manière hétérologue des enzymes fongiques fonctionnelles, nous avons utilisé cet hôte pour la production homologue de la féruloyl esterase-A et pour la production hétérologue du module de fixation à la cellulose du cellulosome de "Clostridium cellulolyticum" (CBM3a), ces deux protéines pouvant jouer un rôle important lors de la délignification. Enfin, nous avons testé la capacité de différentes enzymes de la lygnolyse à délignifier des pâtes à papier de plantes annuelles (pailles de blé et lin) et nous avons pu ainsi démontrer les coopérations existant entre les différentes enzymes lors de la dégradation ciblée de la lignine.AIX-MARSEILLE1-BU Sci.St Charles (130552104) / SudocSudocFranceF

Multiple Correspondence Analysis on Amino Acid Properties within the Variable Region of the Capsid Protein Shows Differences between Classical and Virulent Systemic Feline Calicivirus Strains

Feline calicivirus (FCV) is a widespread and highly prevalent pathogen of domestic cats, responsible for mild upper respiratory tract disease. Outbreaks of severe virulent systemic disease (VSD) associated with FCV infection have been reported worldwide. VSD FCV strains have a broader tropism and cause a systemic vascular compromise. Despite clear differences in the pathogenesis of VSD and oral respiratory infections, attempts to identify specific molecular markers of VSD strains on the major capsid protein VP1 have failed. Region E of VP1 is responsible for the interaction with the cell receptor Junctional Adhesion Molecule JAM-1 (FeJAM-1) and with VP2 minor capsid protein during the entry of the virus. We carried out an original analysis on the sequences from region E of VSD and classical strains. A Multiple Correspondence Analysis was performed on a Boolean matrix built by coding sequences on the basis of their amino acid properties. For the first time, this approach was able to differentiate VSD and classical FCV. Seven remarkable residue positions were shown to be statistically significant for pathotype differentiation, mainly located in the N-terminal hypervariable part of region E. As structural analysis suggested an interaction of these residues with FeJAM-1 or VP2, post-binding events, and specific conformational changes may explain the difference of pathogenesis between pathotypes

Comparison of different fungal enzymes for bleaching high-quality paper pulps

International audienceWild and recombinant hydrolases and oxidoreductases with a potential interest for environmentally sound bleaching of high-quality paper pulp (from flax) were incorporated into a totally chlorine free (TCF) sequence that also included a peroxide stage. The ability of feruloyl esterase (from Aspergillus niger) and Mn2+-oxidizing peroxidases (from Phanerochaete chrysosporium and Pleurotus eryngii) to decrease the final lignin content of flax pulp was shown. Laccase from Pycnoporus cinnabarinus (without mediator) also caused a slight improvement of pulp brightness that was increased in the presence of aryl-alcohol oxidase. However, the best results were obtained when the laccase treatment was performed in the presence of a mediator, 1-hydroxybenzotriazol (HBT), enabling strong delignification of pulps. The enzymatic removal of lignin resulted in high-final brightness values that are difficult to attain by chemical bleaching of this type of pulp. A partial inactivation of laccase by HBT was observed but this negative effect was strongly reduced in the presence of pulp. The good results obtained with the same laccase expressed in A. niger at bioreactor scale, revealed the feasibility of using recombinant laccase for bleaching high-quality non-wood pulps in the presence of a mediator

Structure determination of feline calicivirus virus-like particles in the context of a pseudo-octahedral arrangement.

International audienceThe vesivirus feline calicivirus (FCV) is a positive strand RNA virus encapsidated by an icosahedral T=3 shell formed by the viral VP1 protein. Upon its expression in the insect cell - baculovirus system in the context of vaccine development, two types of virus-like particles (VLPs) were formed, a majority built of 60 subunits (T=1) and a minority probably built of 180 subunits (T=3). The structure of the small particles was determined by x-ray crystallography at 0.8 nm resolution helped by cryo-electron microscopy in order to understand their formation. Cubic crystals belonged to space group P213. Their self-rotation function showed the presence of an octahedral pseudo-symmetry similar to the one described previously by Agerbandje and co-workers for human parvovirus VLPs. The crystal structure could be solved starting from the published VP1 structure in the context of the T=3 viral capsid. In contrast to viral capsids, where the capsomers are interlocked by the exchange of the N-terminal arm (NTA) domain, this domain is disordered in the T=1 capsid of the VLPs. Furthermore it is prone to proteolytic cleavage. The relative orientation of P (protrusion) and S (shell) domains is alerted so as to fit VP1 to the smaller T=1 particle whereas the intermolecular contacts around 2-fold, 3-fold and 5-fold axes are conserved. By consequence the surface of the VLP is very similar compared to the viral capsid and suggests a similar antigenicity. The knowledge of the structure of the VLPs will help to improve their stability, in respect to a use for vaccination

Structural characterization of CA1462, the thiamine pyrophosphokinase-0

In yellow for β-sheets, in red for helices and in green for loops. D1 corresponds to the αβ domain and D2 to the β-sandwich domain. Representation of the B-Factor values on the structure of TPK in complex with thiamine (B) or Thiamine-PNP (C). The tube radius is correlated with B-factors values (high, large tube and small, thin tube). Structures are color coded according to secondary structure elements (red for helices and yellow for β-strands). Ligands are represented in blue. All the structure representations were generated using PYMOL [].<p><b>Copyright information:</b></p><p>Taken from "Structural characterization of CA1462, the thiamine pyrophosphokinase"</p><p>http://www.biomedcentral.com/1472-6807/8/33</p><p>BMC Structural Biology 2008;8():33-33.</p><p>Published online 24 Jul 2008</p><p>PMCID:PMC2515308.</p><p></p

Structural characterization of CA1462, the thiamine pyrophosphokinase-2

coordination of each Mgion in the TPK/thiamine complex (B-C). Fo-Fc electronic density map contoured at 1.5 sigma (displayed in green) of TPNP (D) with Mgand PO4. This map was computed using the refined structure where all ligands have been removed. Coordination of each Mgion in the TPK/thiamine-PNP complex (E-G). Magnesium are represented as white spheres, water molecules as cyan spheres, oxygen atoms are in red, nitrogen in blue and carbon in green. Mgcoordinations are marked with magenta dashed lines.<p><b>Copyright information:</b></p><p>Taken from "Structural characterization of CA1462, the thiamine pyrophosphokinase"</p><p>http://www.biomedcentral.com/1472-6807/8/33</p><p>BMC Structural Biology 2008;8():33-33.</p><p>Published online 24 Jul 2008</p><p>PMCID:PMC2515308.</p><p></p

Structural characterization of CA1462, the thiamine pyrophosphokinase-4

Sed on the mouse TPK (red). Hypothetical AMP is represented with brown lines. B) Stereo view of the 2 alternative conformations of Q138A (gatekeeper) in grey with TPNP (green), Mg(white) and AMP (brown lines) in the potential secondary binding site.<p><b>Copyright information:</b></p><p>Taken from "Structural characterization of CA1462, the thiamine pyrophosphokinase"</p><p>http://www.biomedcentral.com/1472-6807/8/33</p><p>BMC Structural Biology 2008;8():33-33.</p><p>Published online 24 Jul 2008</p><p>PMCID:PMC2515308.</p><p></p