A busca por substâncias benéficas à saúde humana tem impulsionado o desenvolvimento de pesquisas visando o estudo de enzimas e seus produtos através da otimização de bioprocessos. Um dos principais componentes utilizados como base para a indústria de alimentos funcionais são carboidratos denominados frutooligossacarídeos (FOS) derivados da sacarose. Estes são sintetizados por enzimas denominadas frutosiltransferases que podem ser encontradas em plantas, bactérias e fungos. Os FOS têm atraído grande interesse da indústria, devido às suas características fisiológicas e biomoduladoras. Por serem polissacarídeos prebióticos não-digeríveis, têm a capacidade de estimular seletivamente o crescimento de bifidobactérias e lactobacilos, auxiliando na prevenção da cárie dentária e câncer de cólon em humanos. Podem também contribuir na diminuição do colesterol total e triglicerídeos no sangue, promover a reabsorção de cálcio e magnésio e serem utilizados em dietas com restrições alimentares, por serem açúcares de baixo valor calórico e elevado valor nutricional. Tendo em vista a existência de diferentes formas de FOS sintetizados por diferentes mecanismos, o presente trabalho buscou realizar a caracterização estrutural e funcional de um conjunto de 13 frutosiltransferases de bactérias e fungos. Os genes alvo foram clonados e as enzimas expressas e purificadas. Ensaios estruturais e de atividade enzimática, incluindo de hidrólise e polimerização da sacarose, foram conduzidos para a melhor compreensão das bases moleculares envolvidas no reconhecimento do substrato. Oito enzimas, duas β-frutofuranosidases de B. adolescentis, três sucrose-6-phosphate hydrolase de B. licheniformis e L. gasseri e uma invertase de A.niger foram cristalizadas e as enzimas de B. adolescentis e de L. gasseri tiveram suas estruturas resolvidas e seus sítios catalíticos mapeados. Estas apresentam em sua estrutura uma região β-propeller, local identificado como sítio catalítico, conectada à um módulo β-sanduíche. Ambas as enzimas apresentaram atividade hidrolítica da sacarose e a enzima de L. gasseri apresentou a formação dos FOS nistose e 1-cestose com concentrações de 1 M de sacarose bem como em tempos de 8 e 12 horas de incubação. Estes estudos, somados às análises das outras enzimas, permitirão o melhoramento na produção em larga escala, além da otimização e o controle destes processos de obtenção de FOS. / The search for beneficial substances to human health has driven the development of researches on the study of enzymes and their products through bioprocess optimization. One of the main components used as a basis for functional food industry are carbohydrates called fructooligosaccharides (FOS) derived from sucrose. These are synthesized by enzymes called fructosyltransferases which can be found in plants, bacteria and fungi. The FOS has attracted great interest of the industry due to its physiological and biomodulator properties. Because it is non-digestible polysaccharides prebiotics, have the ability to selectively stimulate the growth of bifidobacteria and lactobacilli, assisting in the prevention of tooth decay and colon cancer in humans. They can also contribute to decrease total cholesterol and triglycerides in the blood, to promote the absorption of calcium and magnesium and can be used in diets with dietary restrictions, because they are low-calorie sugars presenting high nutritional value. Considering there are different forms of FOS synthesized by different mechanisms, the present work attempts to make structural and functional characterization of a set of 13 fructosyltransferases of bacteria and fungi. The target genes were cloned and the enzymes were expressed and purified. Structural testing of X-ray crystallography and enzymatic activity, including sucrose hydrolysis and polymerization were carried out for a better understanding of the molecular basis involved in substrate recognition. Eight enzymes, two β-frutofuranosidases B. adolescentis, three sucrose-6-phosphate hydrolase of B. licheniformis and L. gasseri and A. niger invertase, were crystallized and the enzymes from B. adolescentis and from L. gasseri had their structures determined and their catalytic site mapped. These are similar to each other and present in their structure one β-propeller region which was identified as catalytic site, connected to one β-sandwich module. Both enzymes showed hydrolytic activity of the sucrose and L. gasseri showed the formation of FOS, 1-kestose and nystose with 1 M sucrose concentrations and times of 8 and 12 hours of incubation. These studies together with the analysis of other enzymes will enable the improvement in large-scale production, besides the optimization and control of these processes for the production of FOS.

As hidrolases de glicosídeos (GHs) exercem papéis fundamentais em vários processos biomédicos e aplicações industriais. A maioria destas enzimas possui vários domínios funcionais ligados entre si por peptídeos conhecidos como linkers. Informações sobre organização estrutural destas enzimas e sua mobilidade, posições e orientações mútuas de domínios individuais, bem como mudanças conformacionais introduzidas por ligantes ou por mudanças de condições bioquímicas (pH e T) podem ser muito informativas. Por esse motivo, é muito importante determinar a organização estrutural de GHs em termos de posição e orientação de seus domínios individuais e compreender a interação entre estes domínios em condições próximas às fisiológicas. Entretanto, atualmente, a conformação, dinâmica e função dos GHs com múltiplos domínios ainda não são totalmente compreendidas. Assim, o principal objetivo deste projeto foi conduzir estudos de hidrolases de glicosídeos em solução, usando SAS. Um grande número de GHs foi clonado e expresso em laboratório sob a direção do Prof. Dr. Igor Polikarpov (Grupo de Biotecnologia Molecular, IFSC / USP), seguindo protocolos já estabelecidos na literatura, para sua expressão e purificação. Experimentos SAXS foram realizados em colaboração com o Dr. Evandro Ares de Araújo (USP, São Carlos) e com o Prof. Dr. Mário de Oliveira Neto (UNESP, Botucatu). Para estudar as hidrolases de glicosídeos, foi utilizado o método de espalhamento a baixo ângulo, e em adição ao trabalho experimental, foi desenvolvido um novo pacote de software SAXSMoW2 para processar os dados do SAXS. Este pacote permite obter rapidamente os principais parâmetros estruturais de moléculas de proteínas, calcular o peso molecular e o estado oligomérico. Também foi aperfeiçoado e aplicado o método de acoplamento estatístico (statistical coupling analysis) , para complementar os dados estruturais experimentais, em especial para xiloses isomerases. Este método pode permitir uma melhor compreensão da relação entre as características estruturais evolutivas e sua funcionalidade biológica. Além disso, métodos de bioinformática foram desenvolvidos para complementar e compreender melhor as informações estruturais obtidas nos experimentos de SAXS. O primeiro foi um método para separar sequências de GH7 em duas categorias, exo e endogluconases. É útil analisar cada tipo de proteína dentro da família separadamente e estudar o papel dos loops funcionais - características estruturais que influenciam significativamente a atividade biológica. Outro método foi desenvolvido para encontrar o centro de atividade na nova enzima Xilose Isomerase obtida, usando uma estrutura relacionada, bem conhecida, da mesma família. Este método foi aplicado a enzimas cujas estruturas foram estudadas pela técnica de cristalografia em nosso laboratório no IFSC / USP. Inspirado pelo SCA, um método de detecção de comunidades difusas de aminoácidos em proteínas foi desenvolvido. Essa informação também pode complementar os resultados do SCA, indicando conjuntos fortemente correlacionados de aminoácidos na enzima. Outro novo método desenvolvido é uma estimativa de afinidade nas famílias de enzimas ativas em carboidratos utilizando similaridade dos modelos escondidos de Markov e bancos de dados open access de sequências de proteínas. / The Glycoside Hydrolases (GHs) play a key role in a number of biomedical processes and industrial applications. Most of these enzymes are multidomain proteins composed of different functional domains connected by linker peptides. Thus, it is very important to determine structural organization of glycoside hydrolases in terms of positions and orientations of their individual domains and comprehend the interplay between their multiple domains under close-to physiological conditions. To study the glycoside hydrolases, in this work a small-angle scattering method has been used. Currently, the conformation, dynamics and function of GHs with multiple domains are not fully understood. This is why the information on their structural organization and mobility; mutual position and orientation of the individual domains and conformational changes induced by interaction with the substrates or difference in biochemical conditions might be very informative. A large number of GHs have been cloned and expressed in the lab under direction of Prof. Dr. Igor Polikarpov (Molecular Biotechnology group, IFSC/USP) and we follow already established protocols for their expression and purification. SAXS experiments have been carried out in collaboration with Dr. Evandro Ares de Araujo (USP, São Carlos) and Prof. Dr. Mario de Oliveira Neto (UNESP, Botucatu). Additionally to experimental work, a new software package SAXSMoW2 for SAXS data processing has been developed. The software allows to obtain rapidly main structural parameters of the protein molecule, calculate molecular weight and oligomeric state. To supplement an structural data, the method of statistical coupling analysis (SCA) has been significantly improved and applied. The method allows a better understanding of interconnection between evolutionary caused structural features and their biological functionality. Also, various bioinformatic methods were developed to complete and understand better structural information obtained in SAXS experiments. The first one is a method for separating sequences from GH7 into the two bins of exo- and endogluconases. It is helpful to analyze each type of proteins inside the family separately and study the role of functional loops -- structural features that significantly influence on biological activity. Other developed method is for finding of activity center in the new obtained Xylose Isomerase enzyme using related well-known structure from the same family. This method was applied to the enzyme whose structure was studied using crystallography technique in our laboratory at IFSC/USP. Inspired by SCA, a method of aminoacid fuzzy communities detection in proteins has been developed as well. This information also can complete SCA results showing strong correlated sets of aminoacids in the enzyme. Another one new developed method is an estimation of carbohydrate-active family affiliation of unknown proteins using Markov hidden model similarities and open access databanks of protein sequences.

Le contrôle de la glycosylation des protéines recombinantes présente un enjeu considérable dans le développement de la production de protéines d'intérêt thérapeutique dans des systèmes d'expression hétérologues. La glycosylation joue en effet un rôle essentiel dans leurs propriétés, notamment pharmacocinétiques. Le remodelage de la N-glycosylation des protéines recombinantes a été envisagé via l'utilisation de la glycosynthase Cel7B E197A d'Humicola insolens. Cette enzyme doit tout d'abord être modifiée afin de pouvoir fixer un groupement N-acétylglucosaminyle dans le sous-site accepteur +1. Des études de modélisation moléculaire ont mis en évidence deux acides aminés qui pourraient empêcher le positionnement d'une unité glucosidique substituée en position 2. Différents mutants ont été préparés par mutagénèse dirigée afin d'étudier leur spécificité de substrat. Leurs activités glycosynthases ont été caractérisées, montrant l'influence des mutations introduites. Même si la spécificité de substrat désirée n'a pu être obtenue, une modification de la régiosélectivité de la glycosynthase a été mise en évidence vis-à-vis d'un substrat substitué en position 2 par un groupement de type azido.

A busca por substâncias benéficas à saúde humana tem impulsionado o desenvolvimento de pesquisas visando o estudo de enzimas e seus produtos através da otimização de bioprocessos. Um dos principais componentes utilizados como base para a indústria de alimentos funcionais são carboidratos denominados frutooligossacarídeos (FOS) derivados da sacarose. Estes são sintetizados por enzimas denominadas frutosiltransferases que podem ser encontradas em plantas, bactérias e fungos. Os FOS têm atraído grande interesse da indústria, devido às suas características fisiológicas e biomoduladoras. Por serem polissacarídeos prebióticos não-digeríveis, têm a capacidade de estimular seletivamente o crescimento de bifidobactérias e lactobacilos, auxiliando na prevenção da cárie dentária e câncer de cólon em humanos. Podem também contribuir na diminuição do colesterol total e triglicerídeos no sangue, promover a reabsorção de cálcio e magnésio e serem utilizados em dietas com restrições alimentares, por serem açúcares de baixo valor calórico e elevado valor nutricional. Tendo em vista a existência de diferentes formas de FOS sintetizados por diferentes mecanismos, o presente trabalho buscou realizar a caracterização estrutural e funcional de um conjunto de 13 frutosiltransferases de bactérias e fungos. Os genes alvo foram clonados e as enzimas expressas e purificadas. Ensaios estruturais e de atividade enzimática, incluindo de hidrólise e polimerização da sacarose, foram conduzidos para a melhor compreensão das bases moleculares envolvidas no reconhecimento do substrato. Oito enzimas, duas β-frutofuranosidases de B. adolescentis, três sucrose-6-phosphate hydrolase de B. licheniformis e L. gasseri e uma invertase de A.niger foram cristalizadas e as enzimas de B. adolescentis e de L. gasseri tiveram suas estruturas resolvidas e seus sítios catalíticos mapeados. Estas apresentam em sua estrutura uma região β-propeller, local identificado como sítio catalítico, conectada à um módulo β-sanduíche. Ambas as enzimas apresentaram atividade hidrolítica da sacarose e a enzima de L. gasseri apresentou a formação dos FOS nistose e 1-cestose com concentrações de 1 M de sacarose bem como em tempos de 8 e 12 horas de incubação. Estes estudos, somados às análises das outras enzimas, permitirão o melhoramento na produção em larga escala, além da otimização e o controle destes processos de obtenção de FOS. / The search for beneficial substances to human health has driven the development of researches on the study of enzymes and their products through bioprocess optimization. One of the main components used as a basis for functional food industry are carbohydrates called fructooligosaccharides (FOS) derived from sucrose. These are synthesized by enzymes called fructosyltransferases which can be found in plants, bacteria and fungi. The FOS has attracted great interest of the industry due to its physiological and biomodulator properties. Because it is non-digestible polysaccharides prebiotics, have the ability to selectively stimulate the growth of bifidobacteria and lactobacilli, assisting in the prevention of tooth decay and colon cancer in humans. They can also contribute to decrease total cholesterol and triglycerides in the blood, to promote the absorption of calcium and magnesium and can be used in diets with dietary restrictions, because they are low-calorie sugars presenting high nutritional value. Considering there are different forms of FOS synthesized by different mechanisms, the present work attempts to make structural and functional characterization of a set of 13 fructosyltransferases of bacteria and fungi. The target genes were cloned and the enzymes were expressed and purified. Structural testing of X-ray crystallography and enzymatic activity, including sucrose hydrolysis and polymerization were carried out for a better understanding of the molecular basis involved in substrate recognition. Eight enzymes, two β-frutofuranosidases B. adolescentis, three sucrose-6-phosphate hydrolase of B. licheniformis and L. gasseri and A. niger invertase, were crystallized and the enzymes from B. adolescentis and from L. gasseri had their structures determined and their catalytic site mapped. These are similar to each other and present in their structure one β-propeller region which was identified as catalytic site, connected to one β-sandwich module. Both enzymes showed hydrolytic activity of the sucrose and L. gasseri showed the formation of FOS, 1-kestose and nystose with 1 M sucrose concentrations and times of 8 and 12 hours of incubation. These studies together with the analysis of other enzymes will enable the improvement in large-scale production, besides the optimization and control of these processes for the production of FOS.

Orientador: João Alexandre Ribeiro Gonçalves Barbosa / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-08T20:09:24Z (GMT). No. of bitstreams: 1 Valerio_AmandaAbdalla_D.pdf: 3553304 bytes, checksum: 97ba97062f1c23d4b29447c0b9a7316d (MD5) Previous issue date: 2007 / Resumo: Glicosídeo hidrolases (EC 3.2.1.-) são enzimas que hidrolisam ligações glicosídicas. Neste trabalho realizamos estudos funcionais e estruturais de duas glicosídeo hidrolases: a celulase XF0810 de Xylella fastidiosa e a lisozima digestiva 1 de Musca domestica (MdL1). A celulase XF0810 está anotada no genoma da X. fastidiosa como membro da família 5 das glicosídeo hidrolases (EC 3.2.1.4). Após a amplificação, clonagem, expressão e purificação da mesma; prosseguimos com os experimentos de filtração em gel analítica, dicroísmo circular, DLS, ensaio enzimático e cristalização desta proteína. Foi feito um estudo de modelagem onde ficou evidenciado que a XF0810 não pertenceria à família 5 pois quatro dos sete resíduos conservados que caracterizam esta família foram substituídos, incluindo os dois resíduos catalíticos de glutamato essenciais para o mecanismo de clivagem de retenção. Isto foi comprovado através da ausência de atividade nos ensaios feitos com a proteína purificada. A MdL1 pertence à família 22 das glicosídeo hidrolases (EC 3.2.1.17) e foi cristalizada com o ligante Nacetilquitotetraosídeo para a difração de raios X. A resolução da estrutura (2H5Z no PDB) foi realizada por meio do método de substituição molecular tendo como modelo a estrutura nativa. A análise comparativa da MdL1 com outras lisozimas de quatro classes diferentes de animais mostrou grande semelhança e pequenas diferenças apenas na região das voltas. Estas diferenças foram utilizadas para explicar as características especiais de uma lisozima com função digestiva. A volta na região definida pelos resíduos 98-100 apresenta uma deleção na MdL1, tornado-a menos exposta ao solvente, podendo justificar a resistência à proteólise. O resíduo Gln100 participa de uma interação com o ligante. Os resíduos Thr107 e Asn46 são apontados como responsáveis pelo decréscimo dos pKa dos grupos carboxilas dos resíduos catalíticos Glu32 e Asp50, respectivamente. A diminuição dos pKa explica o pH ótimo mais ácido característico de lisozimas digestivas / Abstract: Glycoside hydrolases (EC 3.2.1.-) are enzymes that hydrolyze glycoside bonds. In this work we studied functional and structural features of two glycoside hydrolases: the cellulase XF0810 of Xylella fastidiosa and the digestive lysozyme 1 of Musca domestica (MdL1). The cellulase XF0810 is annotated in the genome of X. fastidiosa as member of family 5 of glycoside hydrolases (EC 3.2.1.4). After the amplification, cloning, expression and purification of XF0810; we continued with the experiments of analytical gel filtration, circular dichroism, DLS, enzymatic assay and crystallization of this protein. A homology model was built which showed that XF0810 did not belong to family 5 because four of seven conserved residues that characterize the family were substituted, including the two catalytic residues of glutamate that are essential for the retention hydrolysis mechanism. This was further confirmed by the absence of activity in the assays (exocellulase with PNPc) performed with the purified protein. The MdL1 belongs to the family 22 of glycoside hydrolases (EC 3.2.1.17) and was crystallized with the ligand N-acetilchitotetraose for X-ray diffraction. The resolution of the structure (2H5Z in PDB) was accomplished by molecular replacement with the native structure as the searching model. The comparative analysis of MdL1 with other lysozymes of four different classes of animals showed a high similarity and few differences appeared only in the loops¿ regions. These differences were used to explain the special characteristics of the lysozyme with a digestive function. The loop in the region defined by residues 98-100 presents one deletion in the MdL1, becoming less exposed to the solvent, this might justify the proteolysis resistance. The residue Gln100 participates directly in an interaction with the ligand. The residues Thr107 and Asn46 are pointed out as responsible for a reduction in the pKa of the carboxyl groups of catalytic residues Glu32 e Asp50, respectively. The reduction in pKa explains the more acidic pH optimum that characterizes the digestive lysozymes / Doutorado / Bioquimica / Doutor em Biologia Funcional e Molecular

Orientador: Fabio Marcio Squina, Roberto Ruller / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-22T02:46:06Z (GMT). No. of bitstreams: 1 Hoffmam_ZairaBruna_M.pdf: 4922807 bytes, checksum: df6edae9e54a10424f07783fcf889151 (MD5) Previous issue date: 2013 / Resumo: O etanol de segunda geração, produzido a partir dos polissacarídeos da biomassa lignocelulósica, apresenta-se como uma alternativa promissora para tornar a matriz energética brasileira sólida e, em grande parte, renovável. A biomassa é composta basicamente por celulose, hemicelulose e lignina. Celulose e hemicelulose devem ser hidrolisadas a açúcares monoméricos fermentescíveis para produzir etanol. Entretanto, a tarefa de propor um processo enzimático de hidrólise eficiente e de baixo custo com a finalidade de sacarificar a biomassa é desafiadora, pois os polissacarídeos que a compõem são complexos e recalcitrantes. Embora as hemiceluloses representem cerca de 30% dos açúcares do bagaço da cana-de-açúcar, uma das biomassas mais promissoras para a produção de etanol de segunda geração no Brasil, ainda poucos estudos são direcionados ao estudo da sacarificação dessa fração de polissacarídeos. Hemicelulases são glicosídeo hidrolases que catalisam a hidrólise dos polissacarídeos hemicelulósicos, e que em atuação sinérgica com celulases promove a hidrólise do bagaço de cana-de-açúcar de maneira mais eficiente que as classes de enzimas isoladamente. A primeira parte dessa dissertação apresenta a caracterização bioquímica e biofísica de uma arabinofuranosidase GH51 do micro-organismo Bacillus subtilis 168. ?-L-arabinofuranosidases são hemicelulases que hidrolisam terminais não redutores de polissacarídeos contendo arabinose. A AbfA mostrou-se robusta, atuando numa ampla faixa de temperatura, e capaz de liberar monômeros de arabinose hidrolisando 1,5-?-L-arabinoheptaose a partir da extremidade não-redutora. A análise dos dados de SAXS combinada com simulações de dinâmica molecular mostrou que a AbfA em solução é hexamérica, com cada subunidade contendo uma molécula com domínio catalítico em enovelado na forma de barril (?/?)8 seguido por um domínio acessório ?-sandwich. Esses dados evidenciam que a estrutura quaternária pode ser importante para o desempenho de atividade das arabinofuranosidases da família 51. A segunda parte da dissertação aborda uma avaliação da influência dos domínios de ligação a carboidratos na atividade de xilanases mesofílicas e termofílicas. Xilanases são hemicelulases que catalisam a hidrólise de ligações endo-1,4-?-D-xilosídicas na molécula de xilano. Fusões gênicas uniram end-to-end um CBM6 de Clostridium thermocellum aos domínios catatalíticos xilanásicos das enzimas GH10 termofílica (de Thermotoga petrophila) e GH11 mesofílica (de Bacillus subtilis 168). O CBM6 alterou o padrão de hidrólise das quimeras termofílicas e aumentou a constante de eficiência da quimera mesofílica, quando comparadas as enzimas nativas respectivas. Além disso, na hidrólise da biomassa da cana-de-açúcar a xilanase quimérica mesofílica suplementou o coquetel comercial Accellerase 1500, liberando mais açúcares redutores. Dessa forma a fusão ao CBM6 mostrou-se uma alternativa para construir enzimas com melhor desempenho cinético / Abstract: The second generation ethanol, produced from lignocellulosic biomass polysaccharides, presents itself as a promising alternative to make the Brazilian energy matrix solid and largely renewable. Biomass is composed mainly of cellulose, hemicellulose and lignin. Cellulose and hemicellulose must be hydrolyzed in fermentable monomeric sugars to produce ethanol. However, the task of proposing a process for enzymatic hydrolysis efficient and low cost in order to saccharify the biomass is challenging because its polysaccharides are complex and recalcitrant, so it requires the presence of several different catalytic activities for hydrolysis. Although hemicelluloses represent about 30% of sugars from sugarcane bagasse, one of the most promising biomass for the production of second generation ethanol in Brazil, few studies are directed to the saccharification study of this polysaccharidic fraction. Hemicellulases are glycoside hydrolases that catalyze the hydrolysis of hemicellulosic polysaccharides and works synergistically with cellulases promoting the hydrolysis of sugarcane bagasse more efficiently than each one class of enzymes alone. The first part of this dissertation presents the biochemistry and biophysics characterization of a GH51 arabinofuranosidase from microorganism Bacillus subtilis 168. ?-Larabinofuranosidases are hemicellulases that hydrolyze linkages in terminal nonreducing ends of polysaccharides containing arabinose. The AbfA showed itself robust, working in a wide temperature range, and able to release arabinose monomers hydrolyzing 1,5-?-L-arabinoheptaose from the non-reducing end. The analysis of SAXS data in combination with molecular dynamics simulations showed that AbfA solution is hexameric, with each subunit containing a molecule with catalytic domain coiled in the form of barrel (?/?)8 followed by a ?-sandwich domain accessory. These results evidence and corroborate data that the quaternary structure may be important for the activity of GH51 arabinofuranosidases family. The second part of the thesis adresses an evaluation of the influence of carbohydratebinding modules, non-catalytic domains of polysaccharides recognition, in the activity of mesophilic and thermophilic xylanases. Xylanases are hemicellulases that catalyze the hydrolysis of endo-1 ,4-?-D-xylosidic linkages in xylan molecule. Gene fusions joined end-to-end a CBM6 from Clostridium thermocellum to catalytic xylanasic domains of enzymes thermophilic GH10 (from Thermotoga petrophila) and mesophilic GH11 (from Bacillus subtilis 168). The CBM6 changed the hydrolysis pattern of thermophilic chimeras increased the efficiency constant of mesophilic chimera, when compared to the respective native enzymes. Furthermore, in the sugarcane biomass hydrolysis mesophilic chimeric xylanase supplemented the commercial cocktail Accellerase 1500, releasing more reducing sugars. In conclusion, the CBM6 fusion proved itself an alternative to construct enzymes with better kinetic performance / Mestrado / Bioquimica / Mestra em Biologia Funcional e Molecular

Orientador: Munir Salomão Skaf / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Química / Made available in DSpace on 2018-08-24T07:03:56Z (GMT). No. of bitstreams: 1 Gomes_ThiagoCostaFerreira_D.pdf: 13438556 bytes, checksum: add103a09a2f775dbe590afd1507f3e4 (MD5) Previous issue date: 2013 / Resumo: Nesta Tese foram investigados sistemas biológicos relacionados à hidrólise da biomassa lignocelulósica. O trabalho de tese está subdividido em três partes. Na primeira parte foram investigadas, mediante simulações por dinâmica molecular, as características estruturais e dinâmicas que conferem termoestabilidade a laminarinases, enzimas da família das glicosideo hidrolases de grande interesse no desenvolvimento de tecnologias viáveis para a produção industrial de etanol celulósico. Na segunda parte do trabalho, desenvolvemos e tornamos publicamente disponível um programa de computador, denominado cellulose-builder, que visa automatizar a construção de arquivos em formato PDB que representem cristalitos de celulose. Os arquivos em formato PDB gerados pelos nosso programa podem ser utilizados como configuração inicial em simulações por dinâmica molecular de sistemas que contenham fases cristalinas da celulose. O programa permite gerar cristalitos de forma e tamanho arbitrários, permitindo expor qualquer face cristalográfica de quatro alomorfos da celulose cristalina. Visando contribuir para o melhor entendimento em nível molecular da estrutura da parede celular vegetal e elucidar os modos de interação entre a celulose e a hemicelulose (dois componentes importantes da parede celular vegetal), na terceira parte deste trabalho empregamos o programa cellulose-builder para construir cristais de celulose com xilanos (hemiceluloses) adsorvidos em diversas faces cristalográficas e utilizamos simulações por dinâmica molecular para estudar em nível atômico a interação entre xilanos e diversas faces cristalográficas do alomorfo I-beta da celulose. Também investigamos o efeito de substituintes comumente presentes em hemiceluloses (acetil e glucuronil) nos parâmetros geométricos e energéticos da interação entre celulose e hemicelulose / Abstract: This Thesis comprises the study of biological systems related to the hydrolysis of lignocellulosic biomass. The work is divided in three parts. In the first part we investigated, by means of molecular dynamics simulations, the structural and dynamical features that confer thermostability to laminarinases, enzymes belonging to the glycoside hydrolase family, which are of great interest in the development of viable tecnologies aiming the industrial production of cellulosic ethanol. In the second part of the Thesis work we developed and made publicly available a computer program, named cellulose-builder, whose purpose is to automatize the construction of PDB-format files representing cellulose crystallites. The PDB-format files generated by our program can be used as starting configurations in molecular dynamics simulations of systems containing crystalline phases of cellulose. The program enables one to generate crystallites of arbitrary size and shape, making it possible to expose any crystallographic face of four cellulose allomorphs. Aiming to contribute to a better understanding of the plant cell wall structure at the molecular level and to elucidate the modes of interaction between cellulose and hemicellulose (two major components of the plant cell wall), in the third part of this work we employed the computer program cellulose-builder to set up cellulose crystals with xylans (hemicelluloses) adsorbed onto several crystallographic faces and applied molecular dynamics simulations to study, at the atomic level, the interactions between xylans and several crystallographic faces of cellulose's allomorph I-beta. We also investigated the effect of substituents commonly occurring in hemicelluloses (namely acetyl and glucuronyl) in geometric and energetic parameters resulting from the interaction between cellulose and hemicellulose / Doutorado / Físico-Química / Doutor em Quimica

Cellulase activity is due to the activity of multiple enzymes, including endoglucanases, cellobiohydrolases and glucosidases that work synergistically to solubilize crystalline cellulose efficiently. The dependence of hydrolysis reaction rate on temperature predicts that large increases in performance and decreased enzyme cost would be achieved if the enzymatic degradation could be operated at elevated temperatures. However there is always a tradeoff between the activity and stability of enzymes. So obtaining cellulases with high thermostability and simultaneously enhanced activity is a great challenge in the field of bioethanol production. In the studies presented in this dissertation, different computational techniques, such as Molecular Dynamics (MD), Molecular Docking, Quantum Mechanics (QM) and hybrid Quantum Mechanics and Molecular Mechanics (QM/MM), along with several site-directed mutagenesis and in vitro assays have been applied to the study and design of the activity and stability of cellulases. Using molecular dynamics to investigate the thermal unfolding of endoglucanases of family 5 of glycoside hydrolases (GH5), a good correlation between the optimum activity temperatures of cellulases and their structural fluctuations was revealed. These data led us to hypothesize that cellulase stability could be enhanced by redesign of enzyme dynamics through altering the amino acid composition in the highly flexible regions of an endoglucanase that would increase its local or global rigidity. Cellulase C, a GH5 member, was stabilized thermally and chemically by cross linking its highly flexible subdomain. Family 1 of glycoside hydrolases were investigated by QM and hybrid QM/MM methods to analyze the role of non-catalytic polar residues at the active site of GH1 glucosidases that make hydrogen bonds to the glucose moiety at subsite -1. A tyrosine residue in simultaneous interaction with O5 of the glucose ring and the carboxylate group of the nucleophilic glutamate was found to play a significant role in the energy profile along the hydrolysis reaction coordinates. It was shown to reduce the energy barrier of the deglycosylation step by ~12 Kcal/mol. Exclusion of this tyrosine from QM calculation substantially influenced the preactivated structure of the glucose moiety in the enzyme-substrate complex and affected the structural distortion and charge distribution in transition states. / Ph. D.

La dégradation des fibres alimentaires est une fonction essentielle des écosystèmes digestifs microbiens. Chez le ruminant, elle est assurée par des bactéries, champignons et protozoaires capables de produire de nombreuses enzymes nécessaires à l’hydrolyse des polysaccharides de paroi végétale. Parmi les facteurs susceptibles d’influencer l’efficacité de dégradation des fibres, qui est une composante importante de la productivité et de la santé animales, des additifs tels que des levures probiotiques apparaissent comme un levier intéressant. Afin d’approfondir les connaissances sur les facteurs de modulation de l’activité fibrolytique, une biopuce ADN fonctionnelle, outil moléculaire haut-débit, ciblant les gènes codant les enzymes clés de la dégradation de la cellulose et des xylanes dans les écosystèmes digestifs a été développée. Aussi, une méthode efficace dédiée à des échantillons ruminaux pour la soustraction des ARNr à partir des ARN totaux a été mise au point afin d’accroitre la sensibilité de l’outil. La biopuce fonctionnelle a été validée sur échantillons de complexité croissante et démontre d’excellents caractères de spécificité et de sensibilité tout en étant exploratoire et quantitative. Des régulations différentielles de l’arsenal des gènes de la fibrolyse de la bactérie du rumen Fibrobacter succinogenes ont pu être montrées. De même, les résultats sur échantillons de rumen suggèrent un rôle des microorganismes eucaryotes dans la fibrolyse pouvant être plus important qu’initialement envisagé. Cette approche métatranscriptomique dirigée pourra in fine continuer d’être appliquée dans l’étude de l’impact de facteurs biotiques et abiotiques sur la fonction fibrolytique microbienne chez les animaux d’élevage. / Dietary fibre degradation is an essential function of microbial digestive ecosystems. In ruminants, this function is ensured by bacteria, fungi and protozoa, producing a large array of enzymes able to degrade plant cell wall polysaccharides. Among factors likely to influence the efficiency of fibre degradation, which is an important component in animal productivity and health, dietary additives such as probiotic yeasts appear as an interesting tool. To provide more insight on factors modulating fibrolytic activity, we designed a functional DNA microarray targeting genes coding for key enzymes involved in cellulose and xylan degradation by digestive microbiota. Also, an efficient method dedicated to rumen samples for removing microorganisms’ rRNA from total RNA samples was developed to increase the sensitivity of the tool. The DNA microarray was validated using targets of increasing complexity and demonstrated sensitivity and specificity as well as explorative and quantitative potential. Differential expression of genes involved in fibrolysis was evidenced in the rumen bacterium Fibrobacter succinogenes. Moreover, results on rumen samples suggest a more important role of eucaryotes in fibre degradation than previously thought. This targeted metatranscriptomic approach will be further applied to the study of the impact of biotic and abiotic factors on the microbial mechanisms of fibre degradation in livestock.

Talaromyces versatilis est un champignon filamenteux d’intérêt industriel grâce à sa capacité deproduction d’enzymes hydrolytiques. La Société Adisseo commercialise un cocktail enzymatiqueproduit par fermentation à partir de T. versatilis, sous le nom de Rovabio™. Ce cocktail est utilisé entant qu'additif alimentaire en nutrition animale, car la grande variété d'enzymes hydrolytiques qu’ilcontient peut dégrader les polysaccharides présents dans l’enveloppe des céréales, améliorant ainsila digestibilité la valeur nutritionnelle des matières premières agricoles. Malgré les efforts consentispour mieux connaître la biologie de T. versatilis, très peu est connu sur ce champignon.L’étudeprésentée ici vise à décrire les réseaux de régulation qui contrôlent l’expression des gènes codantpour ces enzymes hydrolytiques, en utilisant des approches génomiques et transcriptomiques.Avoir accès à une annotation correcte de la séquence génomique et posséder les outilsnécessaires pour l'ingénierie génétique sont essentiels pour réaliser des études de génomiquefonctionnelle. Donc, le premier volet de cette thèse a été l’analyse de la séquence génomique et lacuration manuelle de l'annotation, ce qui nous a conduits à évaluer le vaste potentiel génétique de T.versatilis pour la production et la sécrétion d'enzymes hydrolytiques impliquées dans la dégradationde la lignocellulose. Deuxièmement, un système de délétion des gènes initialement conçu pourAspergillus niger a été adapté à T. versatilis. Cette méthode permet le recyclage du marqueur desélection et est efficace dans des souches dont le système NHEJ est actif (Delmas, et al., 2014, AEM).Au cours de ce travail, deux mutants de délétion de T. versatilis ont été obtenus: ΔxlnR et ΔclrA.La première approche mise en place pour avoir une meilleure compréhension des réseaux derégulation via une vue globale du transcriptome, fut l’utilisation de la technique de RNAseq sur troiséchantillons issus de la souche sauvage de T. versatilis exposée au glucose, à la paille de blé et auglucose et paille de blé simultanément comme sources de carbone, respectivement. Les données ontmontré une augmentation massive des niveaux d’expression de nombreux gènes, en particulier ceuxcodant pour des enzymes hydrolytiques, lorsque le mycélium est exposé à la lignocellulose. Enfin, la dernière partie du projet s’est appuyée sur la la RT-qPCR, technique appropriée pourétudier un nombre limité de gènes dans une grande variété de conditions. Toutefois la normalisationdes données est une étape essentielle du flux de travail qui peut conduire à une interprétationbiologique incorrecte de la régulation des gènes. Le travail effectué sur les données de RNAseq nousa amené à reconsidérer la nature des gènes de référence classiquement utilisés, puisque la plupartd'entre eux présentaient des changements d'expression considérables en présence de lignocellulose.En conséquence, un nouvel ensemble de gènes de référence putatifs a été identifié et la stabilité deleur expression validée par RT-qPCR chez T. versatilis cultivé dans plus de 30 conditions différentes.Des jeux de données de RNAseq de 18 champignons filamenteux phylogénétiquement éloignés ontpar ailleurs été collectés, afin de démontrer que la sélection des gènes candidats pour lanormalisation des données de RT-qPCR chez T. versatilis peut être étendue à d'autres champignons(Llanos et al., 2014, BMC Genomics). Ces aspects méthodologiques validés, nous avons enfin réaliséune étude plus détaillée de la transcription d'un groupe de gènes d'intérêt par RT-qPCR, dans unegrande variété de conditions et 2 souches différentes, la souche sauvage et la mutante ΔxlnR.L'analyse de ces données a permis d'identifier des gènes aux profils d'expression similaires, quirépondent de la même façon aux substrats inducteurs et qui partagent probablement les mêmesmécanismes de régulation. / Talaromyces versatilis is an industrially important enzymes producing filamentous fungus.Adisseo Company commercializes the enzymatic cocktail, produced from T. versatilis fermentation,with the name of Rovabio™. This cocktail is applied as an animal feed additive as it contains a widevariety of hydrolytic enzymes that can degrade the polysaccharides present in the seed-coat and thusimproves the digestibility and increases the nutritional value of the agricultural raw materials.Although efforts have been done to study different aspects of the biology of T. versatilis, very little isknown about this fungus. This study aimed to describe the regulatory networks of genes encodingplant cell wall-degrading enzymes from this biotechnologically important fungus using genomic andtranscriptomic approaches.Having a correct annotation of the genomic sequence together with efficient tools for genomeengineering are essential for downstream functional genomics works and characterization of theregulatory networks. Therefore, the first task carried out an analysis of the genomic sequence and amanual curation of the annotation, which led us to assess the vast genetic potential of T. versatilis forthe production and secretion of hydrolytic enzymes involved in the degradation of lignocellulosicmaterials. Secondly, I adapted a gene deletion system initially designed for Aspergillus niger. Thismethod allows recycling of the selection marker and is efficient in a non-homologous end-joining(NHEJ)-proficient strain (Delmas, Llanos et al., 2014, AEM). During this work, two deletion mutants ofT. versatilis were obtained: ΔxlnR and ΔclrA.Towards better understanding of the regulatory network, I first contributed to an RNAseq-basedtranscriptomic study that was performed on the wild type strain of T. versatilis exposed to glucoseand wheat straw as carbon sources. The data showed a massive increase in transcript levels ofnumerous genes, in particular those encoding hydrolytic enzymes, when the mycelium wasincubated with lignocellulose.If RT-qPCR is indeed a suitable technique to study a limited number of genes in a large variety ofconditions, data normalisation is a critical step of the workflow that can lead to incorrect biologicalinterpretation of gene regulation. The work done on the RNA-seq data led me to reconsider the useof the classical reference genes, since most of them exhibited expression changes in the presence oflignocellulosic substrate. I therefore identified a new set of putative reference genes and validatedtheir expression stability by RT-qPCR in T. versatilis cultivated under more than 30 differentconditions. Then, I collected about a hundred RNA-seq datasets from 18 phylogenetically distantfilamentous fungi, to demonstrate that the use of the suitable candidates for RT-qPCR datanormalisation in T. versatilis can be extended to other fungi (Llanos et al., 2014 BMC genomics (minorrevisions)). Thereafter, I performed a more detailed RT-qPCR based transcriptional study of a groupof genes of interest, in a wide variety of conditions and in 2 strains, the wild-type and the ΔxlnRmutant. The analysis of expression data of the genes of interest allowed to identify genes with similarexpression patterns, which probably share the same regulatory mechanisms and also the substratesthat act as inducers for their expression