Explorando el metagenoma del suelo antártico como fuente de nuevas enzimas adaptadas al frío y elementos génicos móviles

A partir de muestras de suelo antártico se obtuvo la metagenoteca PP1. Esta fue sometida a análisis funcionales y genotípicos para el aislamiento de nuevas enzimas adaptadas al frío con potenciales aplicaciones, y para la detección de elementos génicos asociados a la movilización de genes, respectivamente. Por tamizaje fenotípico se detectaron 14, 14, 3 y 11 clones productores de lipasas/esterasas, proteasas, amilasas y celulasas, respectivamente, con actividades máximas aparentes de 35 °C para las amilasas y lipasas, y de 35-55 °C para las celulasas, tal como se observó para otras enzimas adaptadas al frío. Sin embargo, una celulasa parece ser compatible con enzimas mesófilas, las que usualmente se mantienen activas hasta por sobre 60 °C. Este hecho probablemente esté asociado a un comportamiento psicrotolerante en los suelos antárticos. La metagenómica permite acceder a una nueva miríada de productos metabólicos con potenciales beneficios para aplicaciones biotecnológicas e industriales. Se detectaron los genes tipo intI y tnp por PCR, y sus productos génicos deducidos tuvieron identidades del 58 al 86 % y del 58 al 73 % con secuencias conocidas, respectivamente. Dos clones, BAC 27A-9 y BAC 14A-5, parecen presentar organizaciones sintéticas únicas, lo cual sugiere la existencia de rearreglos génicos probablemente debidos a divergencias evolutivas dentro del género o facilitados por la asociación de elementos de transposición. La evidencia de elementos génicos relacionados con el reclutamiento y la movilización de genes en ambientes extremos como la Antártida refuerza la hipótesis sobre el origen de algunos genes diseminados por elementos móviles entre los microorganismos asociados al ser humano.Metagenomic library PP1 was obtained from Antarctic soil samples. Both functional and genotypic metagenomic screening were used for the isolation of novel cold-adapted enzymes with potential applications, and for the detection of genetic elements associated with gene mobilization, respectively. Fourteen lipase/esterase-, 14 amylase-, 3 protease-, and 11 cellulase-producing clones were detected by activity-driven screening, with apparent maximum activities around 35 °C for both amylolytic and lipolytic enzymes, and 35-55 °C for cellulases, as observed for other cold-adapted enzymes. However, the behavior of at least one of the studied cellulases is more compatible to that observed for mesophilic enzymes. These enzymes are usually still active at temperatures above 60 °C, probably resulting in a psychrotolerant behavior in Antarctic soils. Metagenomics allows to access novel genes encoding for enzymatic and biophysic properties from almost every environment with potential benefits for biotechnological and industrial applications. Only intI- and tnp-like genes were detected by PCR, encoding for proteins with 58-86 %, and 58-73 % amino acid identity with known entries, respectively. Two clones, BAC 27A-9 and BAC 14A-5, seem to present unique syntenic organizations, suggesting the occurrence of gene rearrangements that were probably due to evolutionary divergences within the genus or facilitated by the association with transposable elements. The evidence for genetic elements related to recruitment and mobilization of genes (transposons/integrons) in an extreme environment like Antarctica reinforces the hypothesis of the origin of some of the genes disseminated by mobile elements among "human-associated" microorganisms.Fil: Berlemont, Renaud. Universite de Liege; BélgicaFil: Pipers, Delphine. Universite de Liege; BélgicaFil: Delsaute, Maud. Universite de Liege; BélgicaFil: Angiono, Federico. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología y Biotecnología; ArgentinaFil: Feller, Georges. Universite de Liege; BélgicaFil: Galleni, Moreno. Universite de Liege; BélgicaFil: Power, Pablo. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología y Biotecnología; Argentina. Universite de Liege; Bélgica. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; Argentin

Nueva esterasa tolerante a los solventes orgánicos aislada por metagenómica: ideas sobre la clasificación de las esterasas/lipasas

In order to isolate novel organic solvent-tolerant (OST) lipases, a metagenomic library was built using DNA derived from a temperate forest soil sample. A two-step activity-based screening allowed the isolation of a lipolytic clone active in the presence of organic solvents. Sequencing of the plasmid pRBest recovered from the positive clone revealed the presence of a putative lipase/esterase encoding gene. The deduced amino acid sequence (RBest1) contains the conserved lipolytic enzyme signature and is related to the previously described OST lipase from Lysinibacillus sphaericus 205y, which is the sole studied prokaryotic enzyme belonging to the 4.4 / hydrolase subgroup (abH04.04). Both in vivo and in vitro studies of the substrate specificity of RBest1, using triacylglycerols or nitrophenyl-esters, respectively, revealed that the enzyme is highly specific for butyrate (C4) compounds, behaving as an esterase rather than a lipase. The RBest1 esterase was purified and biochemically characterized. The optimal esterase activity was observed at pH 6.5 and at temperatures ranging from 38 to 45 °C. Enzymatic activity, determined by hydrolysis of p nitrophenyl esters, was found to be affected by the presence of different miscible and non-miscible organic solvents, and salts. Noteworthy, RBest1 remains significantly active at high ionic strength. These findings suggest that RBest1 possesses the ability of OST enzymes to molecular adaptation in the presence of organic compounds and resistance of halophilic proteins.Con el fin de aislar nuevas variantes de lipasas tolerantes a solventes orgánicos (OST), se construyó una librería metagenómica a partir de ADN obtenido de una muestra de suelo de bosque templado. A través de un monitoreo en dos etapas, basado en la detección de actividades, se aisló un clon con actividad lipolítica en presencia de solventes orgánicos. La secuenciación del plásmido pRBest recuperado del clon positivo reveló la presencia de un gen codificante de una hipotética lipasa/esterasa. La secuencia deducida de amino ácidos (RBest1) contiene los motivos conservados de enzimas lipolíticas y está relacionada con la lipasa OST previamente descrita de Lysinibacillus sphaericus 205y, que es la única enzima procariota estudiada perteneciente al subgrupo 4.4 de α/β hidrolasas (abH4.04). Estudios in vivo e in vitro sobre la especificidad de sustratos de RBest1, utilizando triacil-gliceroles o p-nitrofenil-ésteres, respectivamente, revelaron que la enzima es altamente específica para compuestos butíricos (C4 ), comportándose como una esterasa y no como una lipasa. La esterasa RBest1 fue purificada y caracterizada bioquímicamente. La actividad óptima de esterasa fue observada a pH 6,5 y las temperaturas óptimas fueron entre 38 y 45 °C. Se estableció que la actividad enzimática, determinada por hidrólisis de p-nitrofenil ésteres, es afectada en presencia de diferentes solventes orgánicos miscibles y no miscibles, y también sales. Notoriamente, RBest1 permanece significativamente activa a elevadas fuerzas iónicas. Estos hallazgos sugieren que RBest1 posee la capacidad de las enzimas OST de la adaptación molecular en presencia de compuestos orgánicos, así como la resistencia de las proteínas halófilasFil: Berlemont, Renaud. Universite de Liege; BélgicaFil: Spee, Olivier. Universite de Liege; BélgicaFil: Delsaute, Maud. Universite de Liege; BélgicaFil: Lara, Yannick. Universite de Liege; BélgicaFil: Schuldes, Jörg. Universitat of Gottingen; AlemaniaFil: Simon, Carola. Universitat of Gottingen; AlemaniaFil: Power, Pablo. Universite de Liege; Bélgica. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Daniel, Rolf. Universitat of Gottingen; AlemaniaFil: Galleni, Moreno. Universite de Liege; Bélgic

Novel organic solvent-tolerant esterase isolated by metagenomics: insights into the lipase/esterase classification

in order to isolate novel organic solvent-tolerant (oSt) lipases, a metagenomic library was built using dna derived from a temperate forest soil sample. a two-step activity-based screening allowed the isolation of a lipolytic clone active in the presence of organic solvents. Sequencing of the plasmid pRBest recovered from the positive clone revealed the presence of a putative lipase/esterase encoding gene. the deduced amino acid sequence (RBest1) contains the conserved lipolytic enzyme signature and is related to the previously described oSt lipase from Lysinibacillus sphaericus 205y, which is the sole studied prokaryotic enzyme belonging to the 4.4 a/b hydrolase subgroup (abh04.04). Both in vivo and in vitro studies of the substrate specificity of RBest1, using triacylglycerols or nitrophenyl-esters, respectively, revealed that the enzyme is highly specific for butyrate (c4) compounds, behaving as an esterase rather than a lipase. the RBest1 esterase was purified and biochemically characterized. the optimal esterase activity was observed at ph 6.5 and at temperatures ranging from 38 to 45 °c. enzymatic activity, determined by hydrolysis of p‐nitrophenyl esters, was found to be affected by the presence of different miscible and non-miscible organic solvents, and salts. noteworthy, RBest1 remains significantly active at high ionic strength. these findings suggest that RBest1 possesses the ability of oSt enzymes to molecular adaptation in the presence of organic compounds and resistance of halophilic proteins

Etude de l'implication de cellulases dans la voie de biosynthèse de cellulose chez les bactéries

By definition, cellulases are enzymes that catalyze the degradation of cellulose. However, their involvement in cellulose biosynthesis by bacteria and plants has been reported, although their exact contribution remains unclear. In the present study, we have investigated the involvement of cellulases from glycoside hydrolase family 5 (GH5) in cellulose synthesis. In particular, we have functionally and structurally characterized the Ps_Cel5A cellulase from Pseudomonas stutzeri and its metagenome-derived homolog RBcel1, which both belong to the GH5 family and are suspected to be involved in cellulose biosynthesis. In addition, we have also compared these enzymes with the well-characterized Ta_Cel5A cellulase from the cellulolytic fungus Thermoascus aurantiacus. The first part of the work was devoted to the description of the tridimensional structure of RBcel1, in comparison with other glycoside hydrolases from GH5. In the second part of the study, we focused on the functional and structural comparison between RBcel1, Ps_CelA and Ta_Cel5A. Biochemical analysis has highlighted that, besides their hydrolytic activity, RBcel1 and Ps_Cel5A were able to catalyze transglycosylation in vitro. This synthesis reaction was not detected for Ta_Cel5A, which seemed to remain hydrolytic only. Determination of the structure of RBcel1 in complex with cellobiose has revealed distinct features in the aglycone substrate binding sites compared to Ta_Cel5A which could potentially explain the observed differences in their activities in vitro. Finally, the involvement of Ps_Cel5A in cellulose production by P. stutzeri was confirmed, by analysis the ability of P. stutzeri-ΔPs_Cel5A to produce the polymer. Complementation of this mutant strain by the three cellulases was also performed and discussed.Caractérisation de la synthèse de cellulose par deux cellulases d'origine bactérienn

Nouvelle approche pour le Suivi et la Modélisation du comportement au Fluage et du Retrait de Pâtes de Ciment, Mortiers et Bétons depuis la Prise

Lors de la construction de structure réalisée en plusieurs phases de bétonnage, les déformations du béton sont restreintes durant son durcissement. Quand le retrait est restreint, des contraintes de traction sont induites dans le matériau et un risque de fissuration est présent. Il est alors nécessaire de modéliser l’évolution des propriétés au jeune âge afin de prédire le comportement de la structure durant le jeune âge du béton. La difficulté réside dans le fait que la modélisation des propriétés du béton doit être basée sur des données expérimentales au jeune âge et que ces données doivent être obtenues automatiquement car le durcissement du béton se produit rapidement pendant les premières heures et les premiers jours. La thèse porte sur l’étude expérimentale et numérique des propriétés au jeune âge des matériaux à base de ciment et plus particulièrement sur le développement des déformations endogènes, le coefficient de dilatation thermique, le module d’élasticité et le fluage propre en compression et en traction. A cet effet, un travail complet a été réalisé à l'ULB et à l’Ifsttar impliquant le développement d'une nouvelle approche avec de nouvelles procédures d'essai et la conception de nouveaux dispositifs d'essai pour générer des données expérimentales depuis la prise du matériau. La méthodologie est basée sur deux méthodes d'essai répété. Pour la caractérisation du comportement viscoélastique d'un béton depuis sa prise, un essai de chargement permanent couplé à un essai avec des chargements répétés de plusieurs minutes est nécessaire. Les déformations endogènes, le coefficient de dilatation thermique et la prise sont caractérisés avec des variations thermiques répétées sur un échantillon de béton. Cette nouvelle approche a été définie sur un béton ordinaire et ensuite étendue sur l’étude de 4 paramètres pertinents : le rapport eau-ciment, l'effet de restriction de l'agrégat sur la pâte de ciment dans le développement des propriétés du béton au jeune âge, la substitution du ciment par des additions minérales et la différence de comportement en traction et en compression. Sur la base de ces résultats expérimentaux, de nouveaux modèles ont été développés pour la caractérisation des propriétés au jeune âge de matériaux cimentaires depuis le temps de prise. Une version adaptée de la modélisation du fluage propre dans le Code modèle 2010 est également proposéeFor usual concrete structure built in several phases, concrete deformations are restrained during the hardening process. When shrinkage is restrained, tensile stresses are induced and a cracking risk occurs. Modelling the evolution of an early age set of parameters on concrete is necessary to predict the early age behaviour of concrete structures. The difficulty lies in the fact that the modelling of concrete properties must be based on experimental data at early age and this data must be obtained automatically because the hardening process of the concrete takes place rapidly during the first hours and also the first days. The thesis deals with experimental and numerical study of the early age properties of cement based materials and more specifically the development of the autogenous deformation, the coefficient of thermal expansion, the E-modulus and the basic creep in compression and tension. For this purpose, a comprehensive work was carried out at ULB and Ifsttar involving the development of a new approach with new test procedures and the design of new testing devices to generate experimental data since the setting of the material. The methodology is based on two repeated testing methods. For the characterization of the viscoelastic behaviour of a concrete since setting, a permanent loading coupled to a test with repeated minute-long loadings is needed. Whereas, the autogenous strain, the coefficient of thermal expansion and the setting are characterized with repeated thermal variations on a concrete sample. The new approach was defined on an ordinary concrete and then extended to the study of the following parameters: the water-cement ratio, the restrained effect of aggregate on the cement paste in the development of concrete properties at early age, the substitution of cement by mineral addition and the difference of behaviour in tension and in compression. Based on these experimental results, new models were developed for the characterization of the early age properties of cement based materials since setting time. An adapted version of the Model Code 2010 for the modelling of basic creep is also propose

New approach for Monitoring and Modeling of the Creep and Shrinkage behaviour of Cement Pastes, Mortars and Concretes since Setting Time

For usual concrete structure built in several phases, concrete deformations are restrained during the hardening process. When shrinkage is restrained, tensile stresses are induced and a cracking risk occurs. Modelling the evolution of an early age set of parameters on concrete is necessary to predict the early age behaviour of concrete structures. The difficulty lies in the fact that the modelling of concrete properties must be based on experimental data at early age and this data must be obtained automatically because the hardening process of the concrete takes place rapidly during the first hours and also the first days. The thesis deals with experimental and numerical study of the early age properties of cement based materials and more specifically the development of the autogenous deformation, the coefficient of thermal expansion, the E-modulus and the basic creep in compression and tension. For this purpose, a comprehensive work was carried out at ULB and Ifsttar involving the development of a new approach with new test procedures and the design of new testing devices to generate experimental data since the setting of the material. The methodology is based on two repeated testing methods. For the characterization of the viscoelastic behaviour of a concrete since setting, a permanent loading coupled to a test with repeated minute-long loadings is needed. Whereas, the autogenous strain, the coefficient of thermal expansion and the setting are characterized with repeated thermal variations on a concrete sample. The new approach was defined on an ordinary concrete and then extended to the study of the following parameters: the water-cement ratio, the restrained effect of aggregate on the cement paste in the development of concrete properties at early age, the substitution of cement by mineral addition and the difference of behaviour in tension and in compression. Based on these experimental results, new models were developed for the characterization of the early age properties of cement based materials since setting time. An adapted version of the Model Code 2010 for the modelling of basic creep is also proposedLors de la construction de structure réalisée en plusieurs phases de bétonnage, les déformations du béton sont restreintes durant son durcissement. Quand le retrait est restreint, des contraintes de traction sont induites dans le matériau et un risque de fissuration est présent. Il est alors nécessaire de modéliser l’évolution des propriétés au jeune âge afin de prédire le comportement de la structure durant le jeune âge du béton. La difficulté réside dans le fait que la modélisation des propriétés du béton doit être basée sur des données expérimentales au jeune âge et que ces données doivent être obtenues automatiquement car le durcissement du béton se produit rapidement pendant les premières heures et les premiers jours. La thèse porte sur l’étude expérimentale et numérique des propriétés au jeune âge des matériaux à base de ciment et plus particulièrement sur le développement des déformations endogènes, le coefficient de dilatation thermique, le module d’élasticité et le fluage propre en compression et en traction. A cet effet, un travail complet a été réalisé à l'ULB et à l’Ifsttar impliquant le développement d'une nouvelle approche avec de nouvelles procédures d'essai et la conception de nouveaux dispositifs d'essai pour générer des données expérimentales depuis la prise du matériau. La méthodologie est basée sur deux méthodes d'essai répété. Pour la caractérisation du comportement viscoélastique d'un béton depuis sa prise, un essai de chargement permanent couplé à un essai avec des chargements répétés de plusieurs minutes est nécessaire. Les déformations endogènes, le coefficient de dilatation thermique et la prise sont caractérisés avec des variations thermiques répétées sur un échantillon de béton. Cette nouvelle approche a été définie sur un béton ordinaire et ensuite étendue sur l’étude de 4 paramètres pertinents : le rapport eau-ciment, l'effet de restriction de l'agrégat sur la pâte de ciment dans le développement des propriétés du béton au jeune âge, la substitution du ciment par des additions minérales et la différence de comportement en traction et en compression. Sur la base de ces résultats expérimentaux, de nouveaux modèles ont été développés pour la caractérisation des propriétés au jeune âge de matériaux cimentaires depuis le temps de prise. Une version adaptée de la modélisation du fluage propre dans le Code modèle 2010 est également proposé