L’étude des activités enzymatiques exprimées lors de la croissance du champignon de pourriture blanche Trametes versicolor sur milieu malt agar en présence des éprouvettes du hêtre Fagus sylvatica été réalisée. Le processus de dégradation du bois se déroule en deux phases. La première phase est caractérisée par une production d’activités laccases et peroxydases couplées à la dégradation de la lignine. La seconde phase correspond à la production d’activités hydrolytiques impliquées dans la dégradation des polysaccharides. Au début du processus de colonisation du bois, on observe une production d’activités laccases responsables de la dégradation des extractibles du bois, en particulier la catéchine qui est un des composés facilement identifiables dans les extraits du hêtre. La présence d’un biocide comme le propiconazole provoque une forte induction d’activités chitinases lors du stade initial de la colonisation du bois. L’utilisation d’inhibiteurs de chitinases tels que la caféine permet de réduire la quantité du biocide nécessaire pour protéger le bois. La caféine et le rouge congo, un autre agent capable de perturber la mise en place des parois cellulaires du champignon ont un effet additif sur l’inhibition de la croissance de T. versicolor lorsqu’ils sont associés avec le biocide. L’application des méthodes mises en place dans le cadre de notre étude au cas de la dégradation du bois de hêtre traité thermiquement a permis de mettre en évidence l’effet de ce type de traitement sur l’expression des activités enzymatiques expliquant en partie les raisons de l’augmentation de la durabilité du bois suite au traitement thermique. / The study of enzymatic activities expressed during growth of the white rot fungus T. versicolor on malt agar medium in the presence of beech (Fagus sylvatica) wood chips was investigated. The process of wood degradation was divided in two stages. The first stage was characterized by the production of laccases and peroxidases activities. The second stage was characterized by production of carbohydrates hydrolyzing activities. At the beginning of wood colonization process, the production of laccases activities was correlated with degradation of wood acetonic extractives as catechin, which was easily identified in the beech extracts. The presence of a biocide like propiconazole is responsible of the induction of a strong chitinases activity at the beginning of the colonization process. Utilisation of caffeine, a chitinases inhibitor, in the presence of propiconazole allowed to reduce the quantity of biocide necessary to prevent wood from fungal degradation. Similarly, congo red, another cell wall perturbing agent, presented additive effect on inhibition of the fungal growth when associated to propiconazole. The application of the methods developed in this study to the case of heat treated beech wood highlighted the effect of this treatment on the expression of enzymatic activities during wood degradation process explaining in great part the improvement of durability observed after heat treatment.

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Thesis (M.Eng.). / Written for the Dept. of Civil Engineering and Applied Mechanics. Title from title page of PDF (viewed 2008/01/14). Includes bibliographical references.

Título de Ingeniero en Biotecnología Molecular. / La biomasa lignocelulósica es una materia prima renovable prometedora para la producción de combustibles y productos químicos que está disponible a gran escala. Además, la disminución de las reservas de petróleo, junto con el aumento de las emisiones de gases de efecto invernadero, ha dado lugar a la producción y utilización de biocombustibles. En los últimos años, diversos estudios se han centrado en la investigación de diferentes tecnologías para la producción de bioetanol de segunda generación y productos químicos, siendo los bioprocesos basados en hidrólisis enzimática los que se muestran como la opción prometedora. Una etapa crítica del proceso es la conversión de los residuos lignocelulósicos a azúcares fermentables. La bioconversión de la celulosa es catalizada por un grupo de enzimas denominadas celulasas. En trabajos previos se identificó y clonó el gen codificante para una endoglucanasa desde Trametes versicolor (TvEG). El cDNA codificante para la proteína madura fue completamente secuenciado y clonado en el vector pPIC9K, y se expresó como una enzima extracelular activa en la levadura Pichia pastoris KM71. En el presente estudio, como principal objetivo, se purificó y caracterizó TvEG, y se evaluó su potencial hidrolítico en sustratos celulósicos. La cromatografía de intercambio iónico utilizando un FPLC permitió la purificación y concentración de esta enzima desde el sobrenadante del cultivo. Tras análisis mediante SDS-PAGE, se encontró un tamaño molecular mayor a los 42,3 kDa esperados. La hidrólisis catalizada por TvEG en carboximetilcelulosa (CMC) fue máxima a pH 5,6 y 55ºC. La enzima fue estable en un rango de pH de 3 a 9, y hasta los 55ºC; temperatura sobre la cual la estabilidad disminuyó rápidamente después de la incubación durante 15 min. TvEG tuvo la capacidad de liberar azúcares reductores desde papel filtro, Avicel, xilano y paja xiii de trigo. No obstante, las tasas de liberación en comparación a lo obtenido desde CMC fueron bajas, a excepción de lo obtenido desde Avicel (0,0461%) considerando la alta cristalinidad del sustrato. En conclusión, TvEG es una endoglucanasa mesófila. Esta enzima ha demostrado una alta capacidad para liberar azúcares reductores a partir de celulosa cristalina, por lo que podría tener potenciales aplicaciones en la hidrólisis de biomasa lignocelulósica. / Lignocellulosic biomass is a promising renewable feedstock for production of fuels and chemicals that is available at large scale. In addition, the declined oil reserves together with the increased greenhouse gas emissions, has led to the production and use of biofuels. In the last years, several studies have focused on the research of different technologies for the production of second generation bioethanol and chemicals, being enzymatic hydrolysis bioprocesses shown as the promising option. A critical stage in the process is the conversion of lignocellulosic residues into fermentable sugars. The bioconversion of cellulose is catalyzed by a group of enzymes called cellulases. At previous works the gene coding for an endoglucanase (TvEG) from Trametes versicolor was identified and cloned. The cDNA encoding for the mature protein was completely sequenced and cloned into the pPIC9K vector, and expressed as an active extracellular enzyme in the yeast Pichia pastoris KM71 (Salinas et al, 2011). In the present study, as main objective, TvEG was purified and characterized, and its hydrolytic potential in cellulosic substrates was evaluated. Ion exchange chromatography using FPLC allowed the enzyme purification and concentration from the culture supernatant. After SDS-PAGE analysis, a molecular size higher than the 42,3 kDa expected was found. The hydrolysis catalyzed by TvEG in carboxymethyl cellulose (CMC) was maximal at pH 5,6 and 55°C. The enzyme was stable in a pH range of 3 to 9, and up to 55ºC; above which stability decreased rapidly after incubation for 15 minutes. TvEG had the ability to release reducing sugars from filter paper, Avicel and wheat straw. However, the release rates compared to that obtained from CMC were lower, except for Avicel (0,0461%) considering the substrate high crystallinity. xv In conclusion, TvEG is a mesophilic endoglucanase. This enzyme had been shown a high capacity to release reducing sugars from crystalline cellulose, so could have potential applications in the hydrolysis of lignocellulosic biomass

In a limited screen of monokaryons and dikaryons of various strains of Trametes versicolor, the monokaryons generally bleached hardwood and softwood kraft pulps more extensively than dikaryons, although there was great variation among both groups. A monokaryotic strain (52J) derived from a Paprican dikaryon (52P) by regenerating mycelium-derived protoplasts had better pulp bleaching ability than its dikaryotic parent. Unusually low extracellular laccase and manganese peroxidase activity levels were associated with reduced pulp bleaching. Mutants of strain 52J were isolated with various manganese peroxidase and laccase activities. Those with markedly reduced laccase and manganese peroxidase activity had impaired abilities to bleach, delignify, and produce methanol from hardwood kraft pulp, mineralize $ sp{14}$C-labelled synthetic lignins, reduce the colour of kraft mill bleach plant effluent, or had lost these abilities altogether. Dechlorination of kraft bleach plant effluent organochlorines was only slightly less in the mutants tried than in 52J. When purified manganese peroxidase was added back to mutant M49 of 52J, significant pulp bleaching was restored. Effluent decolorization also improved with added manganese peroxidase.

The extracellular culture supernatants of the white rot fungus Trametes versicolor can bleach and delignify unbleached kraft pulps, however the process is too slow for commercial application. Though at least two oxidative enzymes, laccase and manganese peroxidase (MnP) produced by this organism can catalyze a partial delignification of kraft pulp, the effect observed is small relative to that obtained with the complete fungal system. To develop a synthetic (cell-free) delignification system, other protentially important components of the culture supernatant were identified and their contributions to biobleaching and delignification were evaluated. The presence of pulp did not significantly affect the overall carbon balance of the fungus, but a number of non-volatile metabolites (oxalic, fumaric, glyoxylic and phenyllactic acids) induced by the presence of pulp were identified. In T. versicolor 52J, the secretion of manganese peroxidase (MnP), and cellobiose:quinone oxidoreductase (CBQase) enzymes were inducible by pulp whereas cellulase and laccase were not. Several low molecular weight metabolites secreted by T. versicolor functioned as effective Mn(III) complexing agents at their physiological concentrations and promoted MnP activity. / Two distinct CBQase proteins are secreted by T. versicolor 52J, CBQase 4.2 a a 113kDa homodimer containing both heme and flavin cofactors and CBQase 6.4, a 48 kDa monomer with a flavin cofactor only. Superficially, these enzymes appear very similar to the cellobiose oxidase (CBO) and CBQase reported in Phanerochaete chrysosporium. CBQase 4.2 was shown to reduce insoluble manganese dioxide to its soluble Mn(II) and Mn(III) forms with the concommitant oxidation of cellobiose. The sugar acids formed by CBQase could function as effective complexing agents for Mn(III),and complement the Mn(II) to Mn(III) oxidation activity of MnP. / It is proposed that a redox cycling of lignin molecules by certain fungal oxidative and reductive enzymes occurs during delignification and that this cycling ultimately promotes net lignin degradation. A redox cycle wa established between T. versicolor CBQase and laccase which allowed the O$ sb2$ comsumption rate of laccase to remain at a constant level and the total O$ sb2$ consumption by the enzyme was much treater than if the substrate were incubated with laccase alone. A new assay for CBQase based on the ability of this enzyme to reduce the radical intermediates formed during laccase-mediated chlorpromazine oxidation was developed. A redox cycle for these two enzymes was established using both model substrates like CPZ, and with a kraft lignin preparation. CBQase inhibited the formation of polymeric material by laccase; however no evidence was found indicating that cycling with these two enzymes favors depolymerization of kraft lignin. However, the alkali extractability of residual lignin in kraft pulp was increased by a sequential treatment with MnP followed by CBQase.

Although several enzymes presumed to be part of the delignification/kraft pulp biobleaching system of the white-rot basidiomycete Trametes versicolor have been studied, characterized, and even exploited as pulp treatments, the complete system remains poorly understood. Little is known about which enzymes are essential for delignification, how they are regulated, or whether there remain unknown enzymes essential to wood decay in this system. Auxotrophic mutants of T. versicolor 52J were developed and characterized. Plasmid pUC18 was complemented with the T. versicolor 52J genome to create a gene library. Transformation with this plasmid-gene library converted argB and ade2 auxotrophs of T. versicolor 52J to prototrophy. Attempts to rescue the plasmids responsible were unsuccessful. Several different pre-existing plasmid constructs were examined for their potential as selectable markers on the fungus. One of these, pGPhT, worked well at conferring phleomycin resistance. Given that there are now available partial or complete sequences for four T. versicolor laccases, the single cellobiose dehydrogenase, and a lignin peroxidase isozyme, these genetic tools should be very useful in dissecting the mechanisms of white-rot delignification.

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