Orientadora: Profª. Drª. Hana Paula Masuda / Coorientador: Prof. Dr. Danilo da Cruz Centeno / Dissertação (mestrado) - Universidade Federal do ABC, Programa de Pós-Graduação em Biotecnociência, São Bernardo do Campo, 2018. / Os oligossacarídeos da série da rafinose (OSRs) são carboidratos formados pela adição sequencial de um grupo galactosil, geralmente doado por uma molécula de galactinol, à molécula de sacarose. Essa via é regulada principalmente por três enzimas. A galactinol sintase (GOLS) que é responsável pela síntese de galactinol. A rafinose sintase (RAFS) que transfere o resíduo galactosil do galactinol à molécula de sacarose dando origem a rafinose. E a estaquiose sintase (STS) que é responsável pela transferência de galactosil para a rafinose, dando origem a estaquiose. Esses açúcares desempenham importantes papéis fisiológicos nas células vegetais e têm sido considerados como moléculas chave na resposta ao estresse abiótico. Cada enzima envolvida no metabolismo dos OSRs é codificada por uma família de gênica. No entanto, ainda são escassos os trabalhos que apresentem descrições sistemáticas dos genes e suas relações evolutivas nas espécies vegetais. Os poucos trabalhos disponíveis focam nos genes que codificam GOLS, frequentemente considerada a enzima-chave da via. O objetivo deste trabalho foi estudar a diversidade e evolução dos genes rafs e sts em monocotiledôneas, para ampliar o conhecimento sobre os genes nessas espécies. Foram investigados genes rafs e sts em oito espécies vegetais, seis monocotiledôneas e duas dicotiledôneas. Também foram produzidas análises filogenéticas, de ortologia e a caracterização dos domínios proteicos nos genes identificados. Os resultados mostraram que RAFS e STS existem em grande diversidade e que são codificadas por vários genes putativos. As árvores filogenéticas permitiram diferenciar rafs de sts, sugerir relações evolutivas entre os genes e identificar diferentes grupos nessa família gênica. Análises de sintenia indicam a existência de genes ortólogos e duplicações in tandem. Por fim, a análise dos domínios proteicos confirmou a similaridade entre rafs e sts. Como conclusão, essa dissertação expande o conhecimento a respeito dos genes codificadores da via do OSRs, fornece informações para futuros trabalhos com foco em biotecnologia e contribui com a descrição das informações genômicas obtidas nos projetos de sequenciamento genético de espécies vegetais. / The raffinose series oligosaccharides (RFOs) are small carbohydrates synthetized by the sequential addition of a galactosil group, usually donated by a galactinol to sucrose. This metabolic pathway is regulated, among others, by the galactinol synthase (GOLS) enzyme, responsible for the synthesis of galactinol; the raffinose synthase (RAFS), responsible for the transfer of a galactosil group to sucrose, synthetizing rafinose, and; stachyose synthase (STS), responsible for the transfer of another galactosil group to raffinose, thus producing stachyose. These sugars play important physiological roles on plant cells and are considered key molecules in the response to abiotic stress. The enzymes involved on the RFOs metabolism exhibit a large number of functional genes. However, few studies present systematic descriptions of these genes and their evolutionary relationships on plant species. The few available studies focused on the genes that code for GOLS, frequently considered the key enzyme of RFOs metabolic pathway. The objective of this study was to understand the diversity and evolution of the rafs and sts genes in monocot species, to extend the knowledge on these plant genes. Rafs and sts genes were surveyed in eight plant species, six monocot and two dicot species. Phylogenetic and synteny analyses were performed, as well as, the characterization of the protein domains. The results showed that a large number of putative genes codifies both RAFS and STS, indicating that this gene family have a high diversity in plant genomes. The phylogenetic trees allowed proposing the evolutionary relationships between those genes and suggested the existence of different sequence groups. Synteny analyses showed groups of orthologue genes and in tandem gene duplications. Finally, the protein domain analyses corroborated the high similarity between rafs and sts. In conclusion, this work expands the knowledge about RFOs metabolism genes, provided information for further biotechnology studies and contributes to the description of sequence data from genomics projects.

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No description available.

Soybeans are an economically important plant, with an annual crop value that consistently exceeds 20 billion dollars in the United States alone. A recent increase in demand for soybeans, stemming from its diverse applications in products such as animal feed, oil, and biofuel, has created an emphasis for soybean breeders in value added cultivars. These cultivars, have improved, or altered, agronomic or seed composition traits, allowing them to be efficiently utilized in a specific niche of the processing industry. Facilitating the development of such cultivars requires a thorough understanding of the genetic factors that affect the manifestation of value added traits. Value added traits investigated in this study include seed sucrose, raffinose, stachyose, and phytate content, seed weight, and maturity. The objective of the first part of this project was to characterize the source of low seed stachyose in soybean line PI200508. Two F2 populations, developed from PI200508 and soybean introductions which exhibited higher seed stachyose content were utilized in a QTL analysis approach that incorporated the use of the Williams82 whole genome shotgun (WGS) sequence (http://www.phytozome.org) in a candidate gene mapping approach. A predicted soybean galactosyltransferase gene was established as a candidate gene due to its observed segregation with the single low stachyose QTL observed on molecular linkage group (MLG) C2 in both populations. Sequencing of this putative gene revealed a unique 3 bp deletion in PI200508. A marker developed to exploit this deletion accounted for 88% and 94% of the phenotypic variance for seed stachyose content in the two experimental populations, highlighting its potential for use in marker assisted selection of the PI200508 source of low raffinose and stachyose. The second part of this project involved QTL analysis of seed sucrose, raffinose, stachyose, and phytate content, as well as seed weight in a linkage map for a F8 RIL population developed from the Glycine max line V71-370 and the Glycine soja introduction PI40712. Analysis across all 20 soybean MLG identified 25 QTL for these traits on MLG A1, A2, C2, D1b, D2, F, G, H, I, L, M, O. Nine of these QTL were supported across multiple environments, indicating that they, and their associated markers, could be useful to breeders working with these traits. The third part of this project used the same F8 RIL linkage map to investigate time to maturity (Reproductive stage R8). V71-370 and PI407162 differ in time to maturity when grown in Virginia, and the RILs developed from this cross displayed a wide range in maturity. Two major QTL were identified on MLG H and L. Examination of the Williams82 WGS sequence in these QTL regions revealed two predicted genes with homology to Arabidopsis thaliana light response and photoperiodism genes which were investigated as candidate soybean maturity genes. Markers developed from these predicted genes showed close association with the observed QTL, and could facilitate the further investigation of this complex trait. / Ph. D.

Les α-galactosides sont des glucides non digestibles constitués d'unités galactose liées en α(1,6). Les α-galactosides de la famille du raffinose (RFO) sont, avec le saccharose, les principaux oligosaccharides des légumineuses. Cependant, aucune activité α(1,6)-galactosidase n'est retrouvée au niveau de l'épithélium intestinal humain, les RFO sont donc exclusivement fermentés par les enzymes microbiennes. Ces travaux introduisent une enzyme bifonctionnelle de Ruminococcus gnavus E1, un membre majoritaire du microbiote intestinal humain, présentant une activité α(1,6)-galactosidase/ saccharose kinase (AgaSK). L'analyse de la séquence peptidique montre qu'AgaSK présente deux domaines : un domaine homologue aux α-galactosidases GH36, et un autre contenant un motif de fixation des nucléotides (motif A de Walker). La caractérisation des paramètres biochimiques d'AgaSK met en évidence cette bifonctionnalité puisqu'elle est capable d'hydrolyser les α(1,6)-galactosides solubles, et parallèlement en présence d'ATP de phosphoryler le saccharose spécifiquement sur la position C6 du glucose. La production directe de saccharose-6-phosphate à partir de l'hydrolyse du raffinose constitue une voie métabolique jamais décrite chez les bactéries. L'analyse de chacun des domaines montre que les domaines isolés d'AgaSK sont actifs mais la comparaison de leurs paramètres cinétiques montre qu'il y a des différences entre la protéine entière et les domaines isolés. La résolution de la structure du domaine α-galactosidase en complexe avec le galactose démontre que l'état oligomérique est nécessaire pour le bon repliement de la protéine et pour une fixation efficace du substrat. / Α-galactosides are non digestible carbohydrates present in many leguminous plants. Soluble α-galactosides consist of galactose units α(1,6) linked to different carbohydrates. Among these, the raffinose family oligosaccharides (RFO) and sucrose, are the most abundant oligosaccharides found in legumes. However, no α(1,6)galactosidase activity exists in the human intestine mucosa and α-galactosides are exclusively fermented by microbial α(1,6)galactosidases (EC3.2.1.22). Here we introduce a bifunctional enzyme, the α(1,6)galactosidase/sucrose kinase (AgaSK) whose gene is highly transcribed in vivo by Ruminococcus gnavus E1, a major member of human dominant intestinal microbiota. Sequence analysis showed that AgaSK is composed of two domains: one closely related to α-galactosidases from glycoside hydrolase family GH36 and the other containing a nucleotide binding motif (Walker A motif). Its biochemical characterization showed that AgaSK is able to hydrolyze efficiently soluble α-galacosides. Furthermore, AgaSK it is able to bind nucleotide to phosphorylate specifically on the C6 position of glucose sucrose. The production of sucrose-6-P directly from raffinose brings out a glycolytic pathway in bacteria, not described so far. In addition, AgaSK isolated domains are active but the biochemical characterization has shown that there are differences in the activities between the whole protein and isolated domains. The crystal structures of the galactosidase domain in complex with the product shed light onto the reaction and substrate recognition mechanisms and highlight an oligomeric state necessary for efficient substrate binding.

Les agrobactéries établissent des relations à long terme avec les plantes et ce, dans deux styles de vie différents, rhizosphérique et pathogène (galle du collet). Dans ce mode de vie, les bactéries modifient génétiquement leur hôte et se créent ainsi une niche écologique spécifique (tumeur). La transition entre les deux styles de vie est déclenchée par la perception de signaux végétaux, parmi lesquels des acides hydroxycinnamiques (HCAs) comme l’acide férulique. Or dans l’espèce Agrobacterium fabrum, des gènes spécifiques permettent la dégradation des HCAs. Nous avons émis l’hypothèse que cette dégradation était un signal de proximité de la plante et influençait alors des fonctions importantes pour l’interaction avec celle-ci. Nous avons caractérisé la régulation de la dégradation des HCAs, évalué son rôle dans la valeur sélective d’A. fabrum, et suggéré son importance dans la transition entre les styles de vie rhizosphérique et pathogène. Nous avons montré que la dégradation des HCAs module le métabolisme carboné bactérien, notamment l’utilisation d’acide aminés et d’oligosaccharides de la famille du raffinose. Nous avons caractérisé la protéine MelB qui permet l’import de ces sucres, du mélibiose et du galactinol. Leur utilisation est importante pour la colonisation des plantes dès la germination. L’analyse de l’expression des gènes et du métabolisme bactérien en présence d'un composé signal de la plante, nous a révélé de nouveaux déterminants importants pour l’écologie de ce phytopathogène, notamment des facteurs de transcription. En outre, cette analyse a confirmé l’importance des échanges cellulaires et de déterminants impliqués dans la compétition bactérienne / Agrobacterium establish long term interactions with plants, either in a rhizosphere or pathogenic lifestyle. Pathogenic agrobacteria are causing the crown gall disease by genetically modifying the plant cells host, thus creating a specific ecological niche (tumor). The transition from the rhizosphere to the pathogenic lifestyle is triggered by bacterial perception of plant-derived signals, including hydroxycinnamic acids (HCAs) such as ferulic acid. However, A. fabrum strains have species-specific genes that allow HCAs degradation.We hypothesized that in A. fabrum, the degradation of the HCAs is perceived as a plant signal which influences important functions involved in the interaction with plants. We characterized the regulation of HCAs degradation, evaluated its role in the fitness of A. fabrum, and suggested its importance for the transition between the rhizosphere and pathogenic lifestyles. Then, we showed that the degradation of HCAs modulates carbon metabolism, such as the use of amino acids and sugars belonging to the raffinose family oligosaccharides (RFO). We have demonstrated that besides these sugars, the MelB protein allows the import melibiose and galactinol. Their use is important for plant colonization, since seed germination. The analyzes of gene expression and bacterial metabolism in the presence of a plant signal compound, revealed new determinants important for A. fabrum ecology, including transcription factors. In addition, it confirmed the importance of cellular exchanges and bacterial competition for Agrobacterium fitness in planta

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[ES] El tiempo de floración es uno de los caracteres más importantes que influyen en la productividad y el rendimiento de los cultivos. La identificación de compuestos sintéticos que sean bioactivos en el control de la inducción floral es de gran interés. Su identificación podría permitirnos ajustar el tiempo de floración en los cultivos, adaptándolos a las condiciones ambientales más favorables. Para identificar estos compuestos, hemos tomado dos enfoques diferentes: un cribado genético químico y la caracterización del metaboloma de transición floral. En primer lugar, realizamos un rastreo de genética química para identificar moléculas pequeñas que tengan el potencial de controlar la expresión del florígeno, FLOWERING LOCUS T (FT) o la actividad o señalización de FT en Arabidopsis. Para ello, hemos utilizado plantas transgénicas que expresan el gen ß-GLUCURONIDASE (GUS) bajo el control del promotor FT para probar una librería de 360 moléculas preseleccionadas. Los resultados positivos obtenidos se volvieron a analizar mediante un cribado secundario basado en la expresión del gen reportero LUCIFERASE (LUC) bajo el control del promotor FT. Utilizando este enfoque, hemos identificado una molécula que induce con éxito la floración en condiciones de cultivo in vitro. En segundo lugar, hemos caracterizado la función del ácido pipecólico (Pip), una molécula previamente identificada como candidata a regular la floración. Hemos confirmado que las mutaciones en las enzimas responsables de la biosíntesis de Pip muestran una alteración en la respuesta del tiempo de floración. Además, hemos identificado un nuevo papel del Pip relacionado con el crecimiento y el tamaño de la roseta de Arabidopsis. Finalmente, utilizamos un sistema inducible basado en el promotor de CONSTANS (CO) que controla la expresión del gen endógeno de CO fusionado con el receptor de glucocorticoides de rata (CO::GR). De manera que con un solo tratamiento con dexametasona podemos inducir la floración. Con este sistema, realizamos un estudio del metaboloma de muestras de ápices y hojas mediante técnicas de metabolómica dirigida, lipidómica, cuantificación hormonal y transcriptómica. La integración de estos conjuntos de datos ómicos nos ha permitido identificar rutas metabólicas que se encuentran alteradas durante la transición floral. A su vez, la caracterización de mutantes de pérdida de función que codifican enzimas clave de esas vías metabólicas, reveló que algunos de estos mutantes mostraban un fenotipo afectado para el tiempo de floración. Entre ellos, nos enfocamos en la caracterización de los genes relacionados con el metabolismo de la rafinosa, un oligosacárido de reserva. Mutantes afectados en el gen RAFFINOSE SYNTHASE 5 (RS5) presentan un fenotipo de floración temprana y fertilidad reducida. En base a los resultados obtenidos, proponemos un modelo en el que, durante la transición floral, se produce una reestructuración de las ratios entre carbohidratos sencillos (monosacáridos y disacáridos) y de reserva, como la rafinosa. Estos cambios podrían ser modulados por el ácido abscísico (ABA) y por genes relacionados con la floración, desencadenando cambios en el metabolismo de la trehalosa y promoviendo una expresión temprana de FT. / [CA] El temps de floració és un dels caràcters amb més influència en la productivitat i el rendiment dels cultius. La identificació de compostos sintètics bioactius per al control de la inducció floral és de gran interés, ja que la seua identificació podria permetre ajustar el temps de floració dels cultius, aspecte que podria contribuir a l'adaptació a condicions ambientals més favorables. Per a identificar aquests compostos, hem portat a terme dues aproximacions diferents: un garbellat genètic químic i la caracterització del metaboloma de la transició floral. En primer lloc, hem realitzat un cribratge genètic-químicper a identificar xicotetes molècules amb potencial per a controlar l'expressió del florígen, FLOWERING LOCUS T (FT) o l'activitat o la senyalització de FT a Arabidopsis. Per a portar a terme aquest cribratge, hem utilitzat plantes transgèniques que expressen el gen ß-GLUCURONIDASE (GUS) sota el control del promotor de FT amb les quals hem assajat una llibreria de 360 molècules preseleccionades de manera prèvia. Els resultats positius obtinguts en aquest cribratge t s'han sotmés a un cribratge secundari basat en l'expressió del gen reporter LUCIFERASE (LUC) sota el control del promotor FT. La utilització d'aquesta primera aproximació ha permés la idenfiticació d'una molècula que indueix amb èxit la floració en condicions de cultiu in vitro. En En segon lloc, hem caracteritzat la funció de l'àcid pipecòlic (Pip), una molècula prèviament identificada com a candidata a regular la floració. Aquesta aproximació ens ha permet confirmar que mutacions als enzims responsables de la biosíntesi de Pip comporten una alteració al temps de floració. A més, en aquest treball hem identificat un nou paper del Pip relacionat amb el creixement i la grandària de la roseta d'Arabidopsis. Finalment, hem utilitzat un sistema induïble basat en el promotor de CONSTANS (CO) que controla l'expressió del gen endogen de CO fusionat al receptor de glucocorticoides de rata (CO::GR). Aquesta construcció ens proporciona una ferramenta amb la qual induir la floració amb un sol tractament amb dexametasona. A continuació, hem realitzat un estudi del metaboloma de mostres d'àpexs i fulles mitjançant tècniques de metabolòmica dirigida, lipidómica, quantificació hormonal i transcriptòmica. La integració d'aquest conjunt de dades ómiques ens ha permés identificar les rutes metabòliques que es troben alterades durant la transició floral. Al mateix temps, la caracterització de mutants de pèrdua de funció que codifiquen enzims clau per a aquestes rutes metabòliques, ha revelat que alguns d'aquests mutants mostren un fenotip afectat pel que fa al temps de floració. Dintre dels mutants analitzats, ens hem centrat en la caracterització dels gens relacionats amb el metabolisme de la rafinosa, un oligosacàrid de reserva. Els mutants del gen RAFFINOSE SYNTHASE 5 (RS5) presenten un fenotip de floració primerenca i fertilitat reduïda. Sobre la base dels resultats obtinguts, proposem un model en el qual, durant la transició floral, es produeix una reestructuració de les ràtios entre carbohidrats senzills (monosacàrids i disacàrids) i de reserva, com la rafinosa. Aquests canvis podrien ser modulats per l'àcid abscísic (ABA) i per gens relacionats amb la floració, i desencadenariencanvis al metabolisme de la trehalosa, així com la generació de l'expressió primerenca de FT. / [EN] Flowering time is one of the most important traits affecting crop productivity and yield. The identification of natural or synthetic bioactive compounds for the control of flowering induction is of great interest. The identification of compounds with the potential to regulate flowering could allow us to fine-tune flowering responses in crops and adapt them to the changing environmental conditions. To identify these compounds, we have taken two different approaches: a chemical genetic screening and the characterization of the metabolome of floral transition. First, we performed a chemical genetic screening to identify small molecules that have the potential to control the expression of the florigen FLOWERING LOCUS T (FT) or FT activity or signaling in Arabidopsis. We used transgenic plants expressing the ß-GLUCURONIDASE gene (GUS) under the control of the FT promoter to test a preselected library of 360 molecules. Positive hits were retested by a secondary screening based on the expression of the LUCIFERASE (LUC) reporter gene under the control of the FT promoter. Using this approach, we have identified one molecule that successfully induces flowering under in vitro culture conditions. Secondly, we have characterized the function of pipecolic acid (Pip), a molecule previously identified as a candidate to regulate flowering time. We have confirmed that mutations in enzymes responsible for Pip biosynthesis display an altered flowering response. A new role for Pip in rosette growth is also revealed in this work. Finally, we used an inducible system based on the promoter of CONSTANS (CO) driving the expression of CO fused to the rat glucocorticoid receptor (CO::GR). Such a construction provides a tool to induce flowering with a single dexamethasone treatment. We then performed a comprehensive metabolomic study of the shoot apex and leaf samples that included targeted metabolomics, lipidomics, hormone quantification, and transcriptomics. Integration of these omic datasets has allowed us to point out metabolic pathways that are altered during floral induction. Characterization of loss-of-function mutants coding key enzymes of those metabolic pathways revealed that some of these mutants showed a flowering time phenotype. Among them, we focused on the characterization of the contribution of the raffinose metabolism, a storage oligosaccharide, to the determination of flowering time. Mutants affecting RAFFINOSE SYNTHASE 5 (RS5) exhibit an early flowering phenotype and reduced fertility. We propose a model in which the balance between simple and storage carbohydrates in the apex changes during floral induction. This change could be modulated by ABA and flowering-related genes, and it triggers changes in trehalose metabolism, promoting flowering by an early FT upregulation. / Praena Tamayo, J. (2022). Identification of Bioactive Molecules in the Control of Flowering Time [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/185177 / TESIS

Conselho Nacional de Desenvolvimento Científico e Tecnológico / Immobilized enzymes provide many advantages when compared to the usage of their free forms. Among these ones, remarkable advantages are the possibility of the biocatalyst reusability, easy separation at the end of the process, its usage in continuous way and the enhancement of its stability. This work was performed aiming the immobilization of the α-galactosidase enzyme from Aspergillus niger in ion exchange resin and the evaluation of its catalytic activity. Firstly, tests were performed in five different resins: Amberlite 252-Na, Dowex Marathon A, Dowex Marathon C, Duolite A-568 e Duolite S-761. According to the results, Duolite A-568 was chosen as the best support. Therefore, studies were done aiming the optimization of the immobilization process in this resin. Glutaraldehyde 1% (v/v) was used before the enzyme adsorption process and it enhanced the operational stability of the immobilized enzyme. Preliminary tests did not showed difference for the immobilization process at the temperatures of 25 and 40°C. A full factorial design and a central composite design were performed to study the best immobilization conditions varying the pH, the α-galactosidase concentration and the immobilization time. The results led to use the following immobilization conditions: pH 4.5; 15 g/L of α-galactosidase and 3 hours of immobilization. The temperature of maximum activity occurred at 60°C for both free and immobilized enzyme. The activation energy calculated by linear adjustment of Arrhenius equation was 5.66 kcal/mol for soluble α-galactosidase and 4.48 kcal/mol for immobilized α-galactosidase. The optimum pH range obtained for free enzyme was 4.0-5.0 and for immobilized enzyme it was 3.0-6.0. The immobilization process improved the α-galactosidase activity in alkaline pHs. Analysis of pH stability showed that both forms of enzyme were resistant for the pH ranges studied (3.5 to 7.5 for free and 3.0 to 8.0 for immobilized). However, the thermal stability of the biocatalyst immobilized in the support decreased. The kinetic studies without inhibition showed closed values of maximum speed (Vmax) for both enzyme forms (194.5 U for free and 187.7 U for immobilized). Although, the Michaelis-Menten constant (Km) of immobilized enzyme was higher than the free one (18.8 and 12.5 g/L, respectively). The hydrolysis reaction of raffinose was inhibited by the addition of the reaction products, sucrose and galactose, and the results of inhibition by galactose pointed for the competitive inhibition type. Then, storage tests of immobilized α-galactosidase showed that the enzyme maintained its activity even after 145 days when kept at the temperature of 4°C. / O uso de enzimas imobilizadas proporciona muitas vantagens em relação ao seu uso na forma livre. Dentre estas vantagens se destacam a possibilidade de reutilização do biocatalisador, a sua fácil separação ao final do processo, a utilização em modo contínuo e o aumento de sua estabilidade. Este trabalho foi desenvolvido com o objetivo de imobilizar a enzima α-galactosidase de Aspergillus niger em resina de troca iônica e avaliar a sua atividade catalítica. Inicialmente, foram feitos testes preliminares de imobilização em 5 tipos de resinas: Amberlite 252-Na, Dowex Marathon A, Dowex Marathon C, Duolite A-568 e Duolite S-761. Pelos resultados obtidos, Duolite A-568 foi selecionada como melhor suporte e, portanto, estudos foram feitos para a otimização do processo de imobilização nesta resina. Glutaraldeído na concentração de 1% (v/v) foi utilizado anteriormente ao processo de adsorção da enzima e melhorou a estabilidade operacional da α-galactosidase imobilizada. Testes preliminares não indicaram diferença do processo de imobilização para temperaturas de 25 e 40°C. Realizou-se um planejamento fatorial completo e um planejamento composto central para estudar as melhores condições de imobilização variando-se o pH, concentração de α-galactosidase e tempo de imobilização. Os resultados obtidos levaram a utilizar as seguintes condições de imobilização: pH 4,5, concentração de α-galactosidase de 15 g/L e tempo de imobilização de 3 horas. A temperatura de máxima atividade enzimática foi 60°C tanto para a enzima livre quanto imobilizada. O valor da energia de ativação encontrado pelo ajuste linear da equação de Arrhenius foi de 5,66 kcal/mol para α-galactosidase solúvel e 4,48 kcal/mol para α-galactosidase imobilizada. A faixa de pH ótimo obtido para a enzima livre foi 4,0-6,0 e para a enzima imobilizada foi 3,0-6,0. O processo de imobilização melhorou a atividade da α-galactosidase para pHs mais alcalinos. A análise de resistência ao pH mostrou que ambas as formas da enzima foram resistentes para as faixas estudadas (3,5 a 7,5 para livre e 3,0 a 8,0 para imobilizada). No entanto, a resistência térmica do biocatalisador retido no suporte foi menor. O estudo cinético sem inibição apresentou valores de velocidade máxima (Vmáx) próximos para as duas formas da α-galactosidase (194,5 U para livre e 187,7 U para imobilizada), porém o Km da forma imobilizada foi maior que o da livre (18,8 g/L e 12, 5 g/L de rafinose, respectivamente). A reação de hidrólise da rafinose foi inibida pela adição dos produtos da reação, sacarose e galactose, sendo que os resultados de inibição por galactose apontam para o tipo de inibição competitiva Por fim, testes de estocagem da α-galactosidase imobilizada mostraram que a enzima manteve sua atividade mesmo após 145 dias mantida a temperatura de 4°C. / Mestre em Engenharia Química

Orientador: Prof. Dr. Danilo da Cruz Centeno / Coorientadora: Profª. Drª. Hana Paula Masuda / Dissertação (mestrado) - Universidade Federal do ABC, Programa de Pós-Graduação em Biotecnociência, São Bernardo do Campo, 2018. / A busca por fontes renováveis de energia tem aumentado devido à preocupação mundial com o uso excessivo de combustíveis fósseis, destacando-se como alternativa o etanol produzido a partir da cana-de-açúcar. O plantio de cana é intenso no Brasil, por ser uma cultura relativamente bem adaptada às distintas condições locais. No entanto, condições de estresse biótico e abiótico podem reduzir a produtividade e gerar grandes prejuízos, sobretudo em períodos de seca. Nesta perspectiva, o estudo de características agronomicamente desejáveis, como a tolerância ao déficit hídrico, em diferentes variedades é fundamental para a seleção de cultivares apropriadas e o desenvolvimento de cultivares mais produtivas, que elevem a produção de etanol, sem que haja uma expansão demasiada das áreas de cultivo de cana. Neste trabalho, indivíduos de cana-de-açúcar SP80-3280, uma das cultivares mais produzidas no Estado de São Paulo, foram cultivados em condições hídricas distintas e tiveram suas folhas mais jovens (folha +1) analisadas em diferentes aspectos. Os dados fisiológicos foram medidos no Infra-Red GAS Analyzer (IRGA). A quantificação relativa de transcritos foi feita através do método de PCR em tempo real (qPCR), para galactinol sintase (ScgolS), rafinose sintase (ScrafS) e estaquiose sintase (ScstaS), genes-chave na via dos Oligossacarídeos da Série da Rafinose (OSR), envolvida na tolerância à seca. As sequências codificantes de ScgolS, ScrafS e ScstaS, foram obtidas através de análises fenéticas, realizadas com as regiões codificantes de homólogos em milho, sorgo e cana. A quantificação dos metabólitos primários foi realizada através da técnica de cromatografia gasosa acoplada à espectrometria de massas (GC-MS). Plantas que permaneceram sob déficit hídrico por 7 dias (DH - 7 dias) apresentaram diminuição em todos os parâmetros ecofisiológicos, e aumento nos níveis de mio-inositol, manitol, glicose, frutose, ácido quínico e ácido chiquímico. Já plantas com 20 dias de déficit hídrico (DH - 20 dias) mostraram diminuição na taxa de crescimento, no número de folhas verdes e na taxa fotossintética, mesmo com aumento nos níveis de condutância estomática e na concentração interna de CO2. Também apresentaram aumento nos níveis de frutose, galactose, ácido quínico e ácido chiquímico, e redução nos níveis de manitol. As plantas Reidratadas sobreviverem à reidratação e voltaram à crescer após o reestabelecimento da água, apresentando aumento na taxa fotossintética, mesmo com redução nos níveis de condutância estomática, na concentração interna de CO2 e na transpiração. Apresentaram também, aumento nos níveis de mio-inositol, manitol, frutose, ácidos orgânicos e ácidos graxos, e na quantidade relativa de transcritos de ScstaS. Em conclusão, plantas que passaram pelo déficit hídrico apresentaram redução da taxa de crescimento, maior quantidade de compostos relacionados ao ajuste osmótico, e não foi observada indução elevada dos genes-chave da via dos OSR. A sensibilidade de SP80-3280 ao déficit hídrico foi reafirmada, mas foi surpreendente a sobrevivência e reestabelecimento após a reidratação. Aparentemente, SP80-3280 induz mecanismos de prevenção ao déficit hídrico, que possibilitam sua sobrevivência, mas não consegue induzir suficientemente mecanismos de tolerância que garantam à planta melhores condições de crescimento diante do estresse. / The search for renewable sources of energy has increased due to the worldwide preoccupation with the excessive use of fossil fuels, highlighting as alternative the ethanol produced from sugarcane. Cane planting is intense in Brazil, because it is a relatively well adapted crop to the different local conditions. However, biotic and abiotic stress can reduce productivity and generate great losses, especially in periods of drought. In this perspective, the study of agronomically desirable characteristics, such as tolerance to water deficit, in different varieties is fundamental for the selection of appropriate cultivars and the development of more productive cultivars, increasing the ethanol production, without extensive expansion of sugarcane planting areas. In this work, individuals of cultivar SP80-3280, one of the most produced in the State of São Paulo, were cultivated under different water conditions and had their youngest leaves (leaf +1) analyzed in different aspects. Physiological data were measured in the Infra-Red GAS Analyzer (IRGA). The relative quantification of transcripts was done by real-time PCR method (qPCR) for galactinol synthase (ScgolS), raffinose synthase (ScrafS) and stachyose synthase (ScstaS), keygenes in the Raffinose Family Oligosaccharides (RFO), involved in drought tolerance. The coding sequences of ScgolS, ScrafS and ScstaS were obtained through phenetic analyzes performed with the homologous coding regions of maize, sorghum and cane. The primary metabolites were quantified by gas chromatography coupled to mass spectrometry (GC-MS). The plants that remained under water deficit for 7 days (DH - 7 days) presented a decrease in all the ecophysiological parameters, and increased levels of myo-inositol, mannitol, glucose, fructose, quinic acid and shikimic acid. Plants with 20 days of water deficit (DH - 20 days) showed a decrease in the growth rate, in the number of green leaves and in the photosynthetic rate, even with an increase in stomatal conductance and in the internal CO2 concentration. There was also an increase in the levels of fructose, galactose, quinic acid and shikimic acid, and reduction in mannitol levels. The rehydrated plants survived rehydration and returned to grow after water reestablishment, with an increasing in the photosynthetic rate, even with a reduction in stomatal conductance, internal CO2 concentration and transpiration levels. They also showed increased levels of myo-inositol, mannitol, fructose, organic acids and fatty acids, and at the relative amount of ScstaS transcripts. In conclusion, plants that underwent water deficit had a reduction in the growth rate, a greater number of compounds related to the osmotic adjustment, and no high induction of the key genes of the RFO pathway was observed. The sensitivity of SP80-3280 to water deficit was reaffirmed, but survival and reestablishment after rehydration was surprising. Apparently, SP80-3280 induces prevention mechanisms that allow its survival, but it can not sufficiently induce tolerance mechanisms that guarantee the plant better growth conditions in the face of stress.