>Magister Scientiae - MSc / Drought stress is a major contributor to reduced soybean crop yield and quality, this can however be mitigated by the plant’s antioxidant defence mechanisms. One group of antioxidant enzymes that are active in these defence mechanisms are glutathione peroxidases (GPXs). GPXs are antioxidant proteins which are able to reduce H2O2, a toxic reactive oxygen species which accumulates under stress conditions. This study aims at isolating the protein encoded by Glyma01g42840 and determining if it has Phospholipid hydroperoxidase glutathione peroxidase (PHGPX) and/or Thioredoxin dependent peroxidase (TRX-PX) activity as well as assaying the effect of Drought stress on the expression of this putative GPX . This will be accomplished by molecular cloning, sequencing as well as the expression of the isolated protein to assay it enzymatic activity. It was found that the enzyme encoded by Glyma01g42840 is able to use glutathione and thioredoxin as electron donors for the detoxification peroxides, however enzymatic activity is more efficient when using glutathione as an electron donor. In conclusion it was found that glyma01g42840 encodes an enzyme which is able to utilise more than one electron donor and as glutathione produces the greatest amount of enzymatic activity it can be said that glyma01g42840 encodes a GPX.

O-ethyl S-2-diisopropylaminoethyl methylphosphonothiolate (VX) and O-isobutyl-S-2-diethylaminoethyl methylphosphonothioate (R-VX), are considered chemical warfare agents due to their strong acetylcholinesterase-inhibiting properties. Subsequent to terrorist use of these V-type nerve agents in both Japan and the United States of America (the September 11, 2001 attacks) and the limited capability of anti-terrorist groups to detect such weapons, there has been an increased obligation by the Chemical Weapons Convection for specific detection and identification methods for VX and R-VX. Chemical and/or enzymatic hydrolysis yields sulfhydryl mimic products, diethylaminoethanethiol (DEAET) and dimethylaminoethanethiol (DMAET). This thesis investigates the electrocatalytic parameters of DEAET and DMAET using basal plane pyrolytic graphite electrodes (BPPGEs) modified with: (a) single-wall carbon nanotube (BPPGE-SWCNT); (b) SWCNT functionalised with cobalt (II) tetra-aminophthalocyanine by (i) physical (BPPGE-SWCNT-CoTAPc(mix)), (ii) chemical (BPPGE-SWCNT-CoTAPc(cov)) and (iii) electrochemical adsorption (BPPGE-SWCNT-CoTAPc(ads)) processes; (c) nickel powder (BPPGE-Ni); (d) BPPGE-Ni decorated with SWCNT (BPPGE-Ni-SWCNT), and (e) SWCNT functionalised with nickel (II) tetra-aminophthalocyanine (BPPGE-SWCNT-poly-NiTAPc). Electrochemical studies (performed by voltammetric and electrochemical impedance spectroscopic techniques) revealed that the SWCNT and SWCNT-CoTAPc(mix) films showed comparable electrocatalytic responses towards the detection of DEAET and DMAET whereas competitive electrochemical behaviour was seen between SWCNT and SWCNT-NiTAPc modified BPPGEs. Using the BPPGE-SWCNT-CoTAPc(mix), the estimated catalytic rate constants (k) and diffusion coefficients (D) were higher for DEAET than for the DMAET. Also, the detection limits of approximately 8.0 and 3.0µM for DMAET and DEAET were obtained with sensitivities of 5.0×10−2 and 6.0×10−2 AM−1 for DMAET and DEAET, respectively. Unlike BPPGE-SWCNT-CoTAPc(mix) that detected the two sulfhydryls at slightly different potentials, BPPGE-SWCNT did not. The BPPGE-Ni gave enhanced Faradaic response for the redox probe ([Fe(CN)6]3−/4−) and also displayed enhanced electrocatalytic behaviour towards the detection of DMAET and DEAET with high sensitivity (~23x10−3 AM−1) and low detection limits (4.0 – 9.0 µM range). In comparison to other electrodes reported in the literature, BPPGE-Ni exhibits more promising features required for a simple, highly sensitive, fast and less expensive electrode for the detection of the hydrolysis products of V-type nerve agents in aqueous solution. The efficient response of the BPPGE-Ni is attributed to the high microscopic surface area of the nickel powder. The poor response of the BPPGE-Ni-SWCNT suggests that the nickel impurity in SWCNT did not show any detectable impact on the heterogeneous electron transfer kinetics of SWCNT. Unlike the nickel powder, SWCNT and CoTAPc-SWCNT, the NiTAPc-SWCNT hybrid did not show significant electrocatalysis towards the detection of the sulfhydryls. It is interesting, however, to observe for the first time that SWCNT induced crystallinity on the electropolymer of NiTAPc, and that such electropolymer exhibit charge-storage /-transfer properties that greatly enhance the electrochemical response of nitric oxide. / Dissertation (MSc (Chemistry))--University of Pretoria, 2008. / Chemistry / unrestricted

In the forest products industry the opportunity exists to extract currently under-utilised compounds from the process or waste streams and thereby derive more value from the wood entering the process. A big portion of the hemicellulose content of wood does not form part of the final product. Extracting the hemicelluloses from the waste streams or other locations in the process would allow them to be used more effectively. The predominant hardwood hemicellulose, xylan, is polymeric xylose. Xylose is an important platform sugar in bioconversion strategies and can be converted to fuels and other valuable chemicals. The xylan polymer can be hydrolysed to its xylose monomers by a number of conversion strategies; the most widely known being chemical and enzymatic digestion. Chemical conversion is usually done using acid at elevated temperatures, but high yields are often offset by degradation of the product. On the other hand, enzymatic hydrolysis can be better regulated to prevent unwanted degradation of the monomeric sugar products. Enzymatic hydrolysis has been pronounced the environmentally friendly choice of technology, although it is hampered by low conversions and high cost of enzymes. To date commercial enzymes for biomass conversion are not readily available most of which are still in development. In understanding how to best utilise a xylan, recovered from the pulping process, the potential to convert hardwood xylan to xylose with enzymes currently available on the market was studied. A hardwood xylan extracted from fully bleached Eucalyptus pulp with a chelating agent, Nitren, was used as substrate to evaluate the ability of some commercial enzymes to degrade the extracted xylan to xylose monomers. The enzymes used in this study were not dedicated biomass conversion enzymes, but rather chosen for their xylan degrading potential, i.e. xylanase content. By means of hydrolysis profiles on commercial Birchwood and Oat Spelts xylan as substrates and enzyme characterisation, Multifect xylanase was identified as most promising enzyme for xylan conversion. Multifect contained high levels of xylanase and xylosidase activity in the enzyme preparation. Commercial Birchwood xylan and the extracted Eucalyptus xylan were found to be chemically similar, both composed predominantly of xylose. The hydrolysis profiles obtained on Birchwood xylan could therefore serve as a benchmark against which the hy-drolysis of Eucaluptus xylan could be compared. Full conversion of the Eucalyptus xylan with Multifect could not be achieved, although Multifect completely degraded the Birchwood xylan. The maximum xylose yield that could be obtained on Eucalyptus xylan was 80 % and it was concluded that the remaining 20% was unhydrolysable by the enzyme, most likely due to the limitations in the employed extraction method. It was however noted that up to the point of 80 % conversion higher hydrolysis rates were observed on Eucalyptus xylan than Birchwood xylan with equal charges of Multifect. The differences in hydrolysis rates may have indicated that the Eucalyptus xylan is more accessible to enzyme attack than the Birchwood xylan, likely as a result of the extraction methods used to prepare the xylans. A simple economic evaluation illustrated the weight of various costs in process profitability. The most economic operation of a continuous steady state reactor is at a low enzyme charge, 17 IU/ℓ, and a long retention period, five days, due to the high cost of the enzyme compared to other factors. For a reduced retention time, an investigation into enzyme immobilisation and the use of a packed-bed type reactor is recommended. / Dissertation (MEng)--University of Pretoria, 2009. / Chemical Engineering / unrestricted

Natural products continue to be a major chemical lead matter for drug discovery due to their diverse chemical structures and bioactivities. Clinically significant natural products include anti-cancer and anti-infective compounds and while many more of these compounds show promising bioactivity, their clinical relevance is often limited by toxicity or poor solubility. Combinatorial biosynthesis can be employed to modify existing chemical scaffolds towards reducing these limitations. To fully take advantage of these biochemical tools, it is important to understand the biosynthesis and mechanism of action of the molecules. Saccharides in glycosylated natural products provide specific interactions with cellular targets and are often crucial for a compound’s bioactivity. Genetic engineering of sugar pathways can modify glycosylation patterns leading to the diversification of natural products. Saquayamycins (SQN) H and I are cytotoxic angucycline antibiotics containing five deoxyhexoses including the rare amino sugar rednose. Elucidating the biosynthetic pathway of rednose could add to the arsenal of combinatorial biosynthesis tools for drug development. Our research goal of investigating the rednose biosynthetic pathway was pursued through two specific aims: the identification of the Streptomyces sp. KY 40-1 gene cluster involved in the biosynthesis of SQN H and I (sqn) (specific aim 1), and the validation of the proposed L-rednose biosynthetic pathway up to the glycosyl transfer through enzymatic synthesis of NDP-3,6-dideoxy-L-idosamine (specific aim 2). The sqn gene cluster revealed deoxysugar biosynthetic genes that could be used to alter glycosylation patterns to generate novel compounds while the enzymatic synthesis afforded novel genetic engineering tools to generate novel TDP-deoxysugars that could be used to diversify compounds such as aminoglycosides to circumvent resistance mechanisms. The first step to generate TDP-glucosamine enzymatically was accomplished, however later steps were unsuccessful. The aureolic acid mithramycin (MTM) was recently tested in clinical trials for Ewing sarcoma following the discovery of MTM as a potent inhibitor of the oncogenic transcription factor EWS-FLI1 present only in Ewing sarcoma cells It is understood that MTM binds the minor groove of G/C rich DNA as an Mg2+-coordinated dimer disrupting transcription of proto-oncogenes; however, the DNA recognition rules were not completely understood, making further interrogation of MTM’s DNA binding preferences necessary. This research goal of further understanding the mechanism of action for MTM was approached through two specific aims: the investigation of the dimerization of MTM (specific aim 3), and the investigation of MTM’s DNA binding preferences (specific aim 4). This work established that MTM and its biosynthetic precursor premithramycin B (PreMTM B), and several MTM analogues with modified 3-side chains: mithramycin SDK (MTM SDK), mithramycin SA tryptophan (MTM SA-Trp), and mithramycin SA alanine (MTM SA-Ala) dimerize even in the absence of DNA under physiologically relevant conditions. The study also demonstrated that modification of the 3-side chain modulates DNA binding affinity of MTM analogues, established a minimum MTM binding site on DNA, and revealed MTM DNA recognition is driven by direct (sequence) and not indirect (conformation) readout laying the foundation for subsequent research based on the interaction between MTM, DNA, and the oncogenic transcription factor EWS-FLI1 in the rational design of new MTM analogues for the treatment of Ewing sarcoma.

Alarmins and damage associated molecular pattern (DAMP) are endogenous proteins with distinct and various intracellular roles that when released extracellularly act as startingsignals for inflammatory immune responses. The endogenous protein High mobility group box 1 (HMGB1) acts as a DAMP and has been shown to drive progression of multiple inflammatory and autoimmune diseases. During homeostasis HMGB1 is localized in the nucleus of almost any cell, where its main function is organization of the DNA and regulation of transcription. Upon cell death or immune cell activation HMGB1 can be translocated into the cytoplasm for subsequent release into the extracellular space. Extracellular HMGB1 can act as a DAMP by activating several receptors of the immune system. Recent studies focus on HMGB1 release and functional regulation due to prost-translational modifications (PTMs) on cysteine residues. However, little is known about enzymatic regulation of HMGB1. The aim of this thesis was to investigate the possibility of proteolytic processing of HMGB1 by enzymes, which play a crucial role in inflammatory diseases and their progression. We utilized an in vitro model that mimics natural conditions of the autoimmune disease arthritis. Enzymatic digestion of HMGB1 was performed in kinetics studies using the neutrophilic enzymes cathepsin G, neutrophil Elastase as well as matrix metalloproteinase-3, which is released from tissues at the site of inflammation. We defined that HMGB1 is a novel substrate of all of the tested enzymes. All enzymes induced different cleavage pattern. In conclusion, my findings open up the possibility for future studies involving the observed fragments of HMGB1 and their functional features. It also demonstrated that HMGB1 is affected by protease modifications in a disease relevant environment.

La nutrition constitue l'un des facteurs déterminants du développement ou de la prévention de diverses pathologies. Parmi les trois classes de nutriments majeurs, les lipides alimentaires occupent une place essentielle dans cette relation nutrition-santé puisque leur déséquilibre qualitatif est impliqué dans l'incidence des maladies cardiovasculaires. L'importance de l'équilibre alimentaire justifie le développement de produits fonctionnels au profil nutritionnel amélioré. En parallèle, suite à la prise de conscience de l'impact environnemental de la production alimentaire, il est aujourd'hui important d'inscrire le choix des matières premières et des procédés dans une démarche de développement durable.Dans ce contexte, les travaux menés durant cette thèse concernent tout d'abord l'amélioration des propriétés nutritionnelles d'une matière grasse végétale tartinable par réduction de sa teneur en acides gras (AG) saturés délétères, mais également la limitation de son impact environnemental par réduction des ingrédients issus de la filière palme. La mise au point d'un procédé d'interestérification enzymatique vise à ajuster les propriétés rhéologiques du produit sans modifier le profil global en AG, et sans utiliser de solvants ou produits chimiques grâce à l'action d'un biocatalyseur (lipase). Ces travaux ont conduit au développement jusqu'au stade pilote d'un substitut interestérifié par voie enzymatique permettant de réduire la teneur en AG saturés délétères de 65% et l'utilisation de dérivés d'huile de palme de 70% tout en maintenant une fonctionnalité similaire au produit actuel. En parallèle, un procédé enzymatique à également été exploité dans l'étude d'une nouvelle méthode d'analyse de la régiodistribution : l'évaluation des sélectivités de la lipase de Rhizopus oryzae a montré son potentiel pour l'analyse de la position sn-2 des triacylglycérols contenant des AG à chaines moyennes. / Nutrition is one of the main factors contributing to various diseases occurrence or prevention. Among the three major types of nutrients, dietary lipids are essential in this nutrition-health relationship since lipid disorder is associated with an increased risk of cardiovascular diseases. Thus, the importance of a healthy diet explains the development of functional foods with improved nutritional properties. Furthermore, with environmental impact of food production being a growing concern for consumers, selection of raw materials and processes should meet the requirements for sustainable development. In this context, this work concerns the improvement of the nutritional properties of a vegetable oil spread by reducing its unhealthy fatty acids (FA) content, but also the limitation of its environmental impact by minimizing the use of palm oil products. An enzymatic interesterification process has been developed to adapt rheological properties without modifying FA profile nor using solvents and chemical products by action of a biocatalyst (lipase). This work led to the pilot-scale development of an enzymatically interesterified substitute allowing to reduce the unhealthy FA content by 65%, and the use of palm oil products by 70% while keeping a functionality similar to the current product.At the same time, an enzymatic approach has also been used in the investigation of a new method for regiodistribution analysis. Thus, Rhizopus oryzae lipase appeared to be a good candidate for the sn-2 position analysis of triacylglycerols containing medium chain fatty acids.

Ce travail de thèse vise à développer un procédé de synthèse d'esters dans un réacteur enzymatique membranaire fonctionnant en milieu CO2 supercritique. Un tel procédé représente une alternative intéressante à la synthèse chimique classiquement utilisée en industrie car il permet, d'une part, d'utiliser le CO2 supercritique comme solvant et de substituer ainsi les solvants organiques généralement utilisés et, d'autre part, d'avoir un produit final doté d'un label naturel grâce à l'utilisation d'un catalyseur biologique. Dans cette étude, une membrane enzymatique de taille industrielle a été développée. Un pilote spécifique permettant la conduite de réactions enzymatiques en milieu supercritique a été conçu. Le procédé a été testé à l'aide d'une réaction modèle : la synthèse d'acétate d'anisyle à partir d'alcool et d'acétate de vinyle. L'influence de divers paramètres opératoires tels que la température, la pression, ou le débit sur les performances du procédé a été évaluée. Les performances du procédé ont été également comparées à celles d'un réacteur à lit fixe. / This study deals with the development of an enzymatic membrane reactor working in supercritical carbon dioxide for ester synthesis. This process is an alternative to the chemical synthesis classically used in industry because it allows, on the one hand, the use of supercritical carbon dioxide as a solvent instead of organic solvents and on the other hand it leads to natural label ester thanks to the use of a biological catalyst. In this work, an industrial size enzymatic membrane was developed. A special pilot plant was designed to achieve enzymatic reactions in supercritical carbon dioxide medium. The process was studied with a model reaction : the anisyl acetate synthesis from anisic alcohol and vinyl acetate. The impact of several operating conditions like temperature, pressure and flow rate on the process performances was studied. The enzymatic membrane developed in this study was active and showed an interesting conversion rate. The performances were compared to those obtained with a packed bed reactor.

O processo produtivo do couro leva à geração de resíduos sólidos que, por não possuírem boas características de utilização, acabam sendo dispostos em Aterros de Resíduos Industriais Perigosos (ARIP). Por ação microbiológica são degradados lentamente e produzem chorume e biogás (CH4 e CO2), o que pode se prolongar por muitos anos e há necessidade de monitoramento contínuo. Estes resíduos sólidos são em sua maioria farelo de couro wet-blue, um resíduo cromado originário da operação de rebaixamento para padronização e ajuste da espessura de couros, além dos lodos cromados provenientes das estações de tratamento de efluentes dos curtumes. O objetivo deste trabalho foi avaliar a produção de biogás por resíduos de curtume (farelo de rebaixamento e lodo biológico de ETE de curtume), o efeito do tratamento enzimático do farelo de rebaixamento para acelerar sua decomposição e aumentar a produção de biogás e, realizar um levantamento das condições (construção e operação) de ARIPs localizados nas proximidades de Porto Alegre. Os estudos sobre produção de biogás foram avaliados através da realização de experimentos em biorreatores de bacada, utilizando colágeno, farelo de couro wet-blue e lodo com cromo do tratamento biológico de ETE de curtume (inóculo). A avaliação do volume de biogás gerado foi realizada com a utilização de um frasco tipo Mariotte. A composição de metano, dióxido de carbono, oxigênio e nitrogênio foi determinada através de cromatografia gasosa. Os materiais utilizados foram caracterizados quanto ao seu teor de umidade, cinzas, cromo e nitrogênio, e os materiais após o experimento finalizado foram filtrados e caracterizados em relação à massa residual, teor de cromo e nitrogênio. Na primeira série de experimentos foi realizado o prétratamento enzimático térmico para hidrólise do colágeno e resultou em um aumento de 78,3% na produção de biogás e de 76,5% de metano em relação ao colágeno não tratado, porém, a enzima ativa em contato com o inóculo retardou o início da geração de biogás em no mínimo 14 dias. Assim, a inativação da enzima após o tratamento de hidrólise, através de um choque térmico, foi aplicada nos seguintes experimentos utilizando farelo do rebaixamento de couro wet-blue como substrato, resultando para o pré-tratamento enzimático térmico um aumento de 58% na produção de biogás e de 62,4% na produção de metano em relação ao farelo não tratado. Na terceira série de experimentos utilizando somente lodo, foi observado que conforme a quantidade de lodo utilizado é aumentada, o volume de biogás produzido também aumenta. A avaliação do levantamento dos ARIPs foi realizada através da elaboração e aplicação de um questionário com 23 perguntas, a pesquisa mostrou que todos estão em conformidade com a norma brasileira ABNT NBR 10.157/87, que o tipo de resíduo disposto afeta diretamento a produção de chorume e, que tem havido redução da disposição de resíduos de couro wet-blue. / The leather production process leads to generation of solid waste that by not having good characteristics for use, end up disposed of in Hazardous Industrial Waste Landfills (HIWLs). Through microbiological action it’s slowly degraded and produce leachate and biogas (CH4 and CO2), which can last several years and requires continuous monitoring. These solid wastes are mostly wet-blue leather shaving, a chromed waste from the operation of standardizing and to adjust the thickness of the leather, besides the chromed sludge from the effluent treatment system of tanneries. The aim of this study was to evaluate the production of biogas by tannery waste (wet-blue leather shavings and biological sludge from tannery’s WWTP), the effect of enzymatic treatment of the wet-blue leather shavings to accelerate its decomposition and increase biogas production, and conduct the survey about conditions (construction and operational) of HIWLs located near Porto Alegre. Studies on biogas production were evaluated by conducting experiments on bench top bioreactors using collagen, wet-blue leather shavings, chromed sludge from biological treatment of tannery’s WWTP and inoculum. The evaluation of the volume of biogas produced was performed using a Mariotte flask type. The composition of methane, carbon dioxide, nitrogen and oxygen was determined by gas chromatography. The materials used were characterized regarding its moisturet, ash, chromium and nitrogen content, and the materials of the finished experiment were filtered and characterized in relation to the residual mass, chromium and nitrogen content. The first series of experiments carried out the thermal enzymatic pretreatment for hydrolysis of collagen and resulted in a 78.3% increase in biogas production and 76.5% of methane compared to untreated collagen, however, the active enzyme in contact with the inoculum delayed the beginning of biogas generation at least 14 days. Thus, inactivation of the enzyme after hydrolisis treatment, by a heat shock, was applied in the following experiments using wet-blue leather shavings as the substrate, resulting for the thermal enzymatic pretreatment an increase of 58% in the production of biogas and 62.4% in the production of methane compared to untreated leather. In the third series of experiments using only sludge it was observed that as the amount of sludge used increased, the volume of biogas also increased. The evaluation of HIWLs survey was conducted through the development and application of a questionnaire with 23 questions, the survey showed that all are in accordance with the Brazilian standard ABNT NBR 10.157/87, the type of waste disposed directly affects leachate production and there has been a large reduction of disposal of wet-blue leather wastes.

Fruto-oligossacarídeos (FOS) são fibras prebióticas com poder adoçante considerável, sendo um produto de alto valor para a indústria de alimentos. Açúcar invertido é o produto da hidrólise da sacarose possuindo maior poder adoçante, menor susceptibilidade à cristalização e maior higroscopicidade com relação à sacarose, sendo de grande interesse industrial. Ambos produtos podem ser produzidos por reações enzimáticas, utilizando β-frutosiltransferase e β- frutofuranosidase respectivamente, no entanto processos enzimáticos costumam ser caros devido ao alto custo e baixa estabilidade de enzimas. Esses fatores podem ser contornados com a imobilização da enzima, permitindo a reutilização e por vezes aumentando a estabilidade. No presente trabalho a enzima β-frutosiltransferase proveniente de um extrato comercial de Aspergillus aculeatus (Viscozyme L) foi parcialmente purificada, com resina de troca iônica, imobilizada covalentemente em esferas de quitosana e utilizada na produção de FOS. O processo de purificação aumentou a atividade específica em 6 vezes. A estabilidade do biocatalisador imobilizado foi avaliada em 50 bateladas para produção de FOS, foi observado cerca de 55 % de rendimento em cada batelada, sem perda de atividade detectada após as utilizações. Após esse experimento foi testada a utilização das esferas em reatores contínuos com leito fixo e fluidizado, com rendimentos de 59 % e 54 % respectivamente. A produção de açúcar invertido foi feita utilizando a enzima Maxinvert L (β-frutofuranosidase de Saccharomyces cerevisiae) que foi imobilizada, da mesma forma, em esferas de quitosana e sua utilização foi testada em reatores de leito fixo e fluidizado com rendimentos de 98 % e 94 % respectivamente. Os reatores de leito fixo possuem potencial para estudos envolvendo aplicações industriais tanto para produção de FOS quanto para produção de Açúcar Invertido. / Fructooligosaccharides (FOS) are prebiotic fibre with sweetening power, being a highvalue product for the food industry. Invert sugar is the product of sucrose hydrolysis; it has a higher sweetening power, it is less susceptible to crystallization and has a higher hygroscopicity than regular sugar. Finding many uses in food industry processes. Both products can be obtained by enzymatic reactions using β-fructosyltransferase and β- fructofuranosidase, respectively. However, enzymatic processes are often costly because of high enzymatic cost and lack of operational stability. These drawbacks can be overcome by immobilization of enzyme, enabling reuses and usually increasing its stability. In the present work, β-fructofuranosidase from a commercial preparation from Aspergillus aculeatus (Viscozyme L) was partially purified, covalently immobilized on chitosan spheres and used for FOS production. Partial purification resulted in a 6-fold increase in specific activity. Operational stability of biocatalyst was evaluated along 50 batches, resulting in around 55 % yield on each batch and no loss of activity after batches. The immobilized biocatalyst was also used for FOS production in packed bed and fluidized bed reactors with yields of 59 % and 54 % respectively. Invert sugar production was carried out using Maxinvert L (β- fructofuranosidase from Saccharomyces cerevisiae) immobilized, by the same method, on chitosan spheres. Its application on packed bed and fluidized bed reactors was evaluated resulting in yields of 98 % and 94 % respectively. The packed bed reactors presented potential for further studies aiming industrial applications for FOS and Invert Sugar production.

Orientadores: Rubens Maciel Filho, Laura Plazas Tovar / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia Química / Made available in DSpace on 2018-08-27T00:37:21Z (GMT). No. of bitstreams: 1 Lopes_EmiliaSavioli_M.pdf: 7245588 bytes, checksum: 930b1984893789276ff0e09268cf77af (MD5) Previous issue date: 2015 / Resumo: O bagaço da cana-de-açúcar é uma das principais fontes renováveis como alternativa econômica para a produção de biocombustível de segunda geração (2G) e para a geração de eletricidade (cogeração). Neste processo, várias etapas são necessárias, dentre elas, a deslignificação se torna fundamental já que as cadeias celulósicas presentes na lignina dificultam o acesso das enzimas hidrolíticas. Desta forma, o objetivo deste trabalho foi o desenvolvimento, avaliação e o estudo cinético do processo de deslignificação alcalino do bagaço da cana-de-açúcar proveniente da corrente de pré-tratamento com ácido sulfúrico diluído. Foi realizado um pré-tratamento inicial com ácido sulfúrico diluído, seguido de um estudo detalhado da deslignificação com variações de temperatura entre 80 e 120 °C, concentração de NaOH entre 0,5 e 1,5 % m/v e tempo de reação entre 30 e 90 min, estimando os parâmetros cinéticos. Posteriormente, realizou-se a hidrólise enzimática e, em sequência, a fermentação. Os resultados mostram que, com a aplicação do pré-tratamento catalisado com ácido sulfúrico diluído, há uma eficiente conversão das hemiceluloses de 76,31 ± 3,77 % e que temperaturas entre 80 ± 2 °C - 100 ± 2 °C, e tempos de reação de 90 min, mostram-se os mais indicados para o processo de deslignificação, este tendo a maior remoção de lignina encontrada no ponto de 80 °C/0,5 % m/v NaOH/90 min atingindo 75,41 ± 0,73 %. A partir dos dados experimentais, foi possível estimar as constantes de velocidade (kb) constatando-se que estas diminuem com o aumento da temperatura no processo de deslignificação. Com a realização da hidrólise enzimática, foi possível afirmar que após decorridas 24 h ocorreu a liberação máxima dos açúcares, 47,95 g/L no ponto 80 °C/0,5 % m/v NaOH/90 min, decorridas 72 h atingiu-se uma concentração de açúcares liberados de 50,08 g/L e um rendimento global de 53,47 %. Com a realização da fermentação, foram obtidos valores de YP/GLC para o bagaço de cana-de-açúcar PCA e para o seguido de deslignificação de 0,51 kgetanol/kgGLC e 0,47 kgetanol/kgGLC, respectivamente, com seus rendimentos de 100 % e 91,59 %. Vale ressaltar que as fermentações foram realizadas em um reator (PCA) e em um shaker (deslignificado), fator que pode ter levado a obter importantes diferenças nos valores de rendimento / Abstract: Sugarcane bagasse is an important renewable resource with cost-effective alternative for the production of second generation (2G) biofuel and electricity generation (cogeneration). In this process, some steps are required. However, delignification becomes essential due to the cellulose chains present in lignin hinder access of hydrolytic enzymes. Therefore, the goal of this work was the development, evaluation and the kinetic study of alkaline delignification process of sugarcane bagasse from acid pretreatment with dilute sulfuric acid. Firstly, it was carried out the acid pretreatment with dilute sulfuric acid. After, it was developed the detailed study of delignification process. Temperature ranged from 80 to 120 °C, NaOH concentration ranged from 0,5 to 1,5 % m/v and reaction time ranged from 30 to 90 min. Experimental results allowed to estimed the kinetic parameters. Moreover, enzymatic hydrolysis and fermentation processes were carried out. The results show that catalysed pretreatment using dilute sulfuric acid leads an efficient conversion of hemicellulose of 76,31 ± 3,77 % and temperatures between 80 ± 2 °C - 100 ± 2 °C and 90 min establishes the most suitable operating conditions for the delignification process reaching a 75,41 ± 0,73 % of lignin removal at 80 °C/0,5 % w/v NaOH/90 min. Kinetic data were estimated from experimental data. In this sense, the kinetic constants (kb) decrease as the temperature in the delignification process rise. Results from enzymatic hydrolysis showed that the maximum release of sugars occurred after 24 h, 47,95 g/L at 80 °C/0,5 % m/v NaOH/90 min and, at 72 h of enzymatic hydrolysis, the released sugar achieved a concentration of 50,08 g/L and a mass yield of 53,47 %. Results from fermentation process showed that YP/GLC for the PCA sugarcane bagasse and for the followed by delignification of 0,51 kgetanol/kgGLC and 0,47 kgetanol/kgGLC, respectively, attained a process yield of 100 % and 91,59 %, respectively. It is noteworthy that these were carried out in a reactor (PCA) and in a shaker (delignificated), a factor that may have led to obtain important differences in yield values / Mestrado / Desenvolvimento de Processos Químicos / Mestra em Engenharia Química