Pectinases são enzimas que atuam sobre a pectina, um polissacarídeo presente na parede celular vegetal. Estas enzimas são amplamente utilizadas em diversos segmentos industriais, como indústria de alimentos, sucos e vinhos, tecidos, papel, café e óleos, entre outras. As pectinases de uma cepa de Penicillium oxalicum isolada de casca de laranja foram estudadas. Investigamos o efeito na atividade de pectinases de diferentes resíduos agroindustriais como fonte de pectina, o melhor pH e temperatura para estimular a produção de poligalacturonases, pectina liases e pectinesterases. Procedemos a caracterização dos preparados enzimáticos otimizados para as três enzimas e suas aplicações para a extração de sucos de frutas. Investigamos ainda a produção das micotoxinas aflatoxina e ocratoxina. A produção máxima de poligalacturonase foi obtida com P. oxalicum cultivado em meio contendo casca de maracujá. Casca de laranja estimulou a atividade de pectinesterase e pectina liase. Demonstramos ainda que os preparados enzimáticos produzidos por P. oxalicum promovem a extração de sucos, de cor e reduzem a turbidez e a viscosidade dos sucos, tendo alto potencial de aplicação industrial. / Pectinases are enzymes that degraded pectin, a polysaccharide present in plant cell wall. These enzymes are used in different industrial applications, as in the production of juices and wines, degumming tissues, paper, coffee and oil. Pectinases from a strain of Penicillium oxalicum isolated from orange peel was studied. The effect of different agroindustrial residues as source of pectin, the best pH and temperature to improve polygalacturonase, pectin lyase and pectinesterase production where analyzed. We also characterized enzymatic extracts for three enzymes and its application for juice extraction. The production of mycotoxins, alflatoxin and ochratoxin from P.oxalicum were also analyzed. The maximum polygalacturonase production was achieved with P. oxalicum growing in a medium containing passion fruit peel; for pectinesterase and pectin lyase orange peel have highest activity. We demonstrate that enzymatic extracts produced by P. oxalicum promote the extraction of juice, color and reduced the turbidity and viscosity of juices, having industrial application potential.

Pectinases são enzimas que atuam sobre a pectina, um polissacarídeo presente na parede celular vegetal. Estas enzimas são amplamente utilizadas em diversos segmentos industriais, como indústria de alimentos, sucos e vinhos, tecidos, papel, café e óleos, entre outras. As pectinases de uma cepa de Penicillium oxalicum isolada de casca de laranja foram estudadas. Investigamos o efeito na atividade de pectinases de diferentes resíduos agroindustriais como fonte de pectina, o melhor pH e temperatura para estimular a produção de poligalacturonases, pectina liases e pectinesterases. Procedemos a caracterização dos preparados enzimáticos otimizados para as três enzimas e suas aplicações para a extração de sucos de frutas. Investigamos ainda a produção das micotoxinas aflatoxina e ocratoxina. A produção máxima de poligalacturonase foi obtida com P. oxalicum cultivado em meio contendo casca de maracujá. Casca de laranja estimulou a atividade de pectinesterase e pectina liase. Demonstramos ainda que os preparados enzimáticos produzidos por P. oxalicum promovem a extração de sucos, de cor e reduzem a turbidez e a viscosidade dos sucos, tendo alto potencial de aplicação industrial. / Pectinases are enzymes that degraded pectin, a polysaccharide present in plant cell wall. These enzymes are used in different industrial applications, as in the production of juices and wines, degumming tissues, paper, coffee and oil. Pectinases from a strain of Penicillium oxalicum isolated from orange peel was studied. The effect of different agroindustrial residues as source of pectin, the best pH and temperature to improve polygalacturonase, pectin lyase and pectinesterase production where analyzed. We also characterized enzymatic extracts for three enzymes and its application for juice extraction. The production of mycotoxins, alflatoxin and ochratoxin from P.oxalicum were also analyzed. The maximum polygalacturonase production was achieved with P. oxalicum growing in a medium containing passion fruit peel; for pectinesterase and pectin lyase orange peel have highest activity. We demonstrate that enzymatic extracts produced by P. oxalicum promote the extraction of juice, color and reduced the turbidity and viscosity of juices, having industrial application potential.

Pectinases são enzimas que atuam sobre a pectina, um polissacarídeo presente na parede celular vegetal. Estas enzimas são amplamente utilizadas em diversos segmentos industriais, como indústria de alimentos, sucos e vinhos, tecidos, papel, café e óleos, entre outras. As pectinases de uma cepa de Penicillium oxalicum isolada de casca de laranja foram estudadas. Investigamos o efeito na atividade de pectinases de diferentes resíduos agroindustriais como fonte de pectina, o melhor pH e temperatura para estimular a produção de poligalacturonases, pectina liases e pectinesterases. Procedemos a caracterização dos preparados enzimáticos otimizados para as três enzimas e suas aplicações para a extração de sucos de frutas. Investigamos ainda a produção das micotoxinas aflatoxina e ocratoxina. A produção máxima de poligalacturonase foi obtida com P. oxalicum cultivado em meio contendo casca de maracujá. Casca de laranja estimulou a atividade de pectinesterase e pectina liase. Demonstramos ainda que os preparados enzimáticos produzidos por P. oxalicum promovem a extração de sucos, de cor e reduzem a turbidez e a viscosidade dos sucos, tendo alto potencial de aplicação industrial. / Pectinases are enzymes that degraded pectin, a polysaccharide present in plant cell wall. These enzymes are used in different industrial applications, as in the production of juices and wines, degumming tissues, paper, coffee and oil. Pectinases from a strain of Penicillium oxalicum isolated from orange peel was studied. The effect of different agroindustrial residues as source of pectin, the best pH and temperature to improve polygalacturonase, pectin lyase and pectinesterase production where analyzed. We also characterized enzymatic extracts for three enzymes and its application for juice extraction. The production of mycotoxins, alflatoxin and ochratoxin from P.oxalicum were also analyzed. The maximum polygalacturonase production was achieved with P. oxalicum growing in a medium containing passion fruit peel; for pectinesterase and pectin lyase orange peel have highest activity. We demonstrate that enzymatic extracts produced by P. oxalicum promote the extraction of juice, color and reduced the turbidity and viscosity of juices, having industrial application potential.

Computational tools play an important role in the description of biological systems. Scientists describe and study structure, conformational changes and interactions between molecules in silico, often as a cheaper and faster alternative for biosynthesis. The simulated dynamic behavior in time of a molecular system is a straight forward source of information about substrate-enzyme interactions at the atomic level, and a powerful tool for the identification of molecular properties important in enzymatic reactions. Our study is focused on the computational investigation of structure and substrate specificity of hydrolases important in biotransformation. The computational work was performed in close collaboration with biochemists-experimentalists from Charles University and the Microbiological Institute of the Academy of Sciences of the Czech Republic. Hydrolases have great a potential in the chemoenzymatic synthesis of modified carbohydrates with regulated properties. Carbohydrates, as substrates of hydrolases, are important in normal functionality of many organisms. They have a dual role in immune response regulation: some carbohydrates (like GlcNAc and ManNAc) participate in activation and some (like GalNAc) in suppressing immunity; glycosidase deficiency is associated with a number of lysosomal disorders. We used homology modeling, computational docking and molecular dynamics simulation (MD) methods for the complex study of fungal hydrolases: alpha-galactosidase/alpha-N-acetylgalactosaminidase from Aspergillus niger; beta-N-acetylhexosaminidases (HEX) (from Aspergillus oryzae and Penicillium oxalicum); nitrilase from Aspergillus niger. Our structural study unambigously demonstrates that the enzyme encoded by genes variant A (aglA) from A. niger is able to accept alpha-N-acetylgalactosamine as its substrate and explains structural features responsible for its specificity. Homology models of HEXs from P. oxalicum and A. oryzae were built and compared. Homology models were used to study the role of protein glycosylation, disulfide bonds, dimer formation and interaction with natural and modified substrates. Model of nitrilase from Aspergillus niger helped to analyze multimer formation.

in English β-N-Acetylhexosaminidase (EC 3.2.1.52) is exoglycosidase, which exhibits the unique properties in the filamentous fungi. Enzyme from these organisms are dimeric, inducible and secreted extracelluary. It is expresed as preproprotein, consists of a signal sequence, a large propeptid and a catalytic subunit. Although the enzyme is widely distributed, its structure differs in varies organisms. Bacteria have only monomeric hexosaminidase. Human enzymes are dimeric as well as fungal, but only hexosaminidase from filamentous fungi have the catalytic subunit noncovalently associated with the propeptide. Propeptide is a essential for the enzyme activity. It exists a homologues model of the catalytic subunit of β-N-acetylhexosaminidase from Penicillium oxalicum, but the structure of the propeptide has not yet been solved. The first part of this diploma thesis deals with the optimization of production and purification conditions. The second part deals with structural studies of β-N-acetylhexosaminidases from the filamentous fungi Penicillium oxalicum CCF 3438. These studies were carried out using chemical cross-linking and high resolution mass spectrometry. The combination of these methods revealed region of the noncovalent interaction of the catalytic subunit with the propeptide.

L'objectif de la présente thèse a été d'étudier les possibilités de valorisation des déchets de biomasse (Penicillium oxalicum var. Armeniaca, Tolypocladium sp.) issus de procédés de fermentation en tant que biosorbants du plomb, du cadmium, du mercure et du chrome hexavalent. Alors que la biomasse de Tolypocladium (biomasse T) a été utilisée en l'état, la biomasse de Penicillium a dû subir un traitement alcalin (biomasse P3). Dans un premier temps, la biosorption a été étudiée à partir de solutions synthétiques monométalliques. Le pH initial optimal pour les espèces cationiques est proche du pH neutre, alors que celui de l'espèce anionique est inférieur à 3 (milieu acide). Le biosorbant P3 a montré des capacités de fixation plus importantes pour tous les métaux étudiés par rapport au biosorbant T. Les capacités maximales de fixation peuvent atteindre, dans des conditions opératoires optimales, 270 mg Hg g-1 biomasse P3 (à pH 5), 160 mg Hg g-1 biomasse T (à pH 7), 70 mg Cr(VI) g-1 biomasse P3 (à pH 3) et 55 mg Cr(VI) g-1 biomasse T (à pH 2). Ces valeurs figurent parmi les valeurs les plus élevées citées dans la littérature. Au contraire pour le plomb et le cadmium les performances sont décevantes. La cinétique de biosorption du mercure et du chrome est du second ordre. L'influence des phénomènes de diffusion sur la vitesse globale du procédé a été largement discutée. Pour les espèces cationiques, le mécanisme de chélation des métaux sur les groupes aminés a été proposé pour la biomasse P3, alors que l'échange d'ions sur les groupes carboxyliques s'est révélé plus probable pour la biomasse T. Pour ce qui concerne les espèces anioniques (ions chromates) la fixation procède par attraction electrostatique sur les groupes aminés protonés. Concernant la biomasse T, le mécanisme combiné d'adsorption/réduction observé pendant les expérimentations en régime statique n'a pas été clarifié. La biosorption à partir de solutions mixtes a été également étudiée. La sélectivité de la biomasse P3 pour le mercure a été confirmée. Aucun mécanisme de compétition n'a été identifié. Le travail expérimental s'est conclu par des expérimentations en régime dynamique (colonne à lit fixé) en utilisant la biomasse de Tolypocladium, immobilisée sur une terre des diatomées. Le procédé est contrôlé par diffusion intraparticulaire. Les courbes de percée dépendent de plusieurs paramètres tels que la concentration initiale et le diamètre des particules.