Thesis (MSc (Biochemistry))--University of Stellenbosch, 2011. / ENGLISH ABSTRACT: Due to a global lack of access to potable water, a problem particularly affecting people in developing countries and the poor, improvement on existing water purification methods are necessary to provide more cost effective, accessible and efficient methods of water purification. In drinking water systems, biofilms are a potential source of contamination, which can affect the biological stability and hygienic safety of water. In industrial water systems, biofilms can cause corrosion, resistance in flow systems and a decrease in efficiency of membranes. Nanotechnology has been identified as a technology to utilize in water purification problem solving. Alternatives to the use of chemical biocides and antibiotics need to be investigated therefore; the focus of this study was the fabrication and characterization of polymer nanofibers containing silver nanoparticles as biocide and anti-biofouling nanofibers with hydrolytic enzymes immobilized on the surface. The aim of this study was to synthesize and compare poly (vinyl alcohol) (PVA) nanofibers and poly (acrylonitrile) (PAN) nanofibers with silver nanoparticles to determine which type of fiber will be the most appropriate for application in water sanitation. The two types of fibers were to be compared based on morphology, silver nanoparticle content, physical distribution of silver nanoparticles, levels of silver leaching from the fibers in water, which could imply toxicity, and most importantly, anti-microbial efficacy. Back scattering electron images revealed that silver nanoparticles in PVA nanofibers were more evenly dispersed than in PAN nanofibers, but that PAN nanofibers had higher silver nanoparticle content. This was confirmed by energy dispersive X-ray (EDX) analysis. Both PVA and PAN nanofibers containing silver nanoparticles had excellent anti-microbial activity, with PVA nanofibers killing between 91% and 99% of bacteria in a contaminated water sample and PAN nanofibers killed 100%. When investigated by SEM, the biocidal effect of PAN nanofibers containing silver nanoparticles can be observed as morphological changes in the cell walls. Neither PVA nor PAN nanofibers leached silver into water. PVA is a non-toxic and biodegradable synthetic polymer, and PVA-silver nanofibers have excellent anti-microbial activity, making it applicable in water sanitation in an environmental conscious milieu. PAN nanofibers are more conductive to the formation of silver nanoparticles, have higher silver nanoparticle content, allowing the complete sanitation of pathogenically contaminated water samples. PAN nanofibers also have better longevity and strength in water, making it ideal for water filtration and sanitation in higher throughput systems. Furthermore, immobilized enzymes are being investigated as possible alternatives to inefficient conventional methods of controlling and removing biofilms from filtration systems. This study demonstrates the covalent immobilization of two industrial proteases and an amylase enzyme onto polymer nanofibers widely used in filtration membranes. Confirmed by FTIR, these nanofibers were successfully activated by amidination, allowing the covalent immobilization of respectively two serine proteases and an α-amylase onto the fibers. When inspected visually, fibers largely retained their original morphology after activation and enzyme immobilization. Immobilized enzymes were, however visible as aggregated particles on the nanofiber surfaces. The large surface area to volume ratio provided by the nanofibers as immobilization surface, allowed sufficient amounts of enzymes to be immobilized onto the fibers so that all enzymes retained above 80% of the specific activity of the free enzymes. For each of the immobilized enzymes, just below 30% of initial activity was retained after 10 repeated cycles of use. Fibers with immobilized enzymes on their surface did not support the growth of biofilms, as opposed to plain nanofibers, which did support the growth of biofilms. When considering the combined advantages of this effective immobilization process, the robustness of the enzymes used in this study, and their effectiveness against biofilms in their immobilized state, a valuable addition has been made to technology available for the control of biofilm formation on filtration membranes, and could potentially be employed to control biofilm formation in water filtration systems. A combination of anti-microbial and anti-biofouling nanofibers into a single nanofiltration product may prove to be highly applicable in water sanitation systems. / AFRIKAANSE OPSOMMING: As gevolg van 'n wêreldwye gebrek aan toegang tot drinkbare water, 'n probleem wat veral mense in ontwikkelende lande en armes raak, is dit van belang dat bestaande metodes van watersuiwering verbeter word om voorsiening te maak vir meer koste-effektiewe, toeganklike en doeltreffende metodes van watersuiwering. In drinkwater stelsels is biofilms 'n potensiële bron van besoedeling, wat die biologiese stabiliteit en die higiëniese veiligheid van water beïnvloed. In industriële waterstelsels kan biofilms tot die verwering van pyplyne lei, weerstand in die stroomstelsels veroorsaak en 'n afname in die doeltreffendheid van membrane veroorsaak. Nanotegnologie is geïdentifiseer as 'n tegnologie wat aangewend kan word in watersuiwerings probleemoplossing. Alternatiewe vir die gebruik van chemiese antimikrobiese middels moet dus ondersoek word. Hierdie studie fokus dus op die vervaardiging en karakterisering van polimeer nanovesels met silwer nanopartikels wat ingesluit is as antimikrobiese middel en anti-biofilm vesels met hidrolitiese ensieme geïmmobiliseer op die oppervlak. Die doel van hierdie studie was om poli (viniel alkohol) (PVA) nanovesels en poli (akrielonitriel) (PAN) nanovesels te sintetiseer waarby silwer nanopartikels ingesluit is, en te bepaal watter tipe vesel die mees geskikte sal wees vir die gebruik in water sanitasie. Die twee tipes vesels is met mekaar vergelyk gebaseer op morfologie, silwer nanopartikel inhoud, fisiese verspreiding van silwer nanopartikels, vlakke van silwer uitloging vanuit die vesels in water, wat toksisiteit tot gevolg kan hê, en die belangrikste, antimikrobiese effektiwiteit. Terug verstrooiing elektron beelde het aan die lig gebring dat die silwer nanopartikels in PVA nanovesels meer eweredig versprei was as in PAN nanovesels, maar dat PAN nanovesels 'n hoër silwer nanopartikel inhoud gehad het. Dit is bevestig deur “energy dispersive X-ray” (EDX) analise. Beide PVA en PAN nanovesels met silwer nanopartikels het uitstekende antimikrobiese aktiwiteit getoon, met PVA vesels wat tussen 91% en 99% bakterieë in besoedelde water monsters kon doodmaak en PAN vesels wat 100% bakterieë kon uitwis. Wanneer vesels ondersoek is met ʼn skandeer elektronmikroskoop (SEM), kon die antimikrobiese effek van PAN vesels met silwer nanopartikels as morfologiese veranderinge in die selwande waargeneem word. Nie PVA of PAN nanovesels loog silwer uit in water nie. PVA is 'n nie-toksiese en bioafbreekbare sintetiese polimeer, en PVA-silwer nanovesels het uitstekende antimikrobiese aktiwiteit, wat dit van toepassing maak op water sanitasie in ʼn omgewings bewuste milieu. PAN vesels is meer gunstig tot die vorming van silwer nanopartikels, en het 'n hoër silwer nanopartikel inhoud, dus word patogeen besoedelde water volledig gesteriliseer. PAN vesels het ook 'n beter langslewendheid en weerstandige sterkte in water, wat dit ideaal vir water filtrasie en sanitasie in hoër deursettings stelsels maak. Geïmmobiliseerde ensieme word ook ondersoek as moontlike alternatiewe tot ondoeltreffende konvensionele metodes van beheer en die verwydering van biofilms uit water stelsels. Hierdie studie toon die kovalente immobilisasie van twee industriële proteases en 'n amilase ensiem op polimeer vesels wat gebruik word in filtrasie membrane. Bevestig deur FTIR, is PAN vesels suksesvol geaktiveer deur amidinasie, sodat die kovalente immobilisasie van onderskeidelik twee serien proteases en 'n α-amilase op die vesels moontlik is. Met visuele ondersoek kan gesien word die vesels behou grootliks hul oorspronklike morfologie na aktivering en ensiem immobilisasie. Geïmmobiliseerde ensieme is egter sigbaar as saamgevoegde deeltjies op die nanovesel oppervlaktes. Die groot oppervlakarea: volume-ratio van die vesels wat dien as immobilisasie oppervlak, laat toe dat voldoende hoeveelhede van ensieme geïmmobiliseer word sodat alle ensieme meer as 80% van die spesifieke aktiwiteit van die vrye ensieme behou. Vir elk van die geïmmobiliseer ensieme, is net minder as 30% van die aanvanklike aktiwiteit behou na 10 siklusse van hergebruik. Vesels met geïmmobiliseerde ensieme op hul oppervlaktes het nie die groei van biofilms ondersteun nie, in teenstelling met gewone vesels, sonder ensieme, wat die groei van biofilms ondersteun. As die gesamentlike voordele van hierdie doeltreffende immobilisasie proses, die robuustheid van die ensieme en hulle doeltreffendheid teen biofilms in hul geïmmobiliseerde toestand in ag geneem word, is ʼn waardevolle toevoeging gemaak tot tegnologie wat beskikbaar is vir die beheer van biofilm vorming op filtrasie membrane, en dit kan potensieel gebruik word om biofilm vorming filter stelsels te beheer. Die kombinasie van anti-mikrobiese en anti-biofilm vesels in ʼn enkele nanofiltrasie produk moet nagestreef word, omdat dit hoogs van toepassing sal wees in water sterilisasie stelsels.

Thesis (MSc)--University of Stellenbosch, 2011. / ENGLISH ABSTRACT: In this thesis the synthesis of a wide range of model and siloxane functionalized N-(n-propyl)-1-(2-pyridyl and quinolyl)-imine ligands (L1-L6) are described. Functionalized ligands (L4-L6) were obtained by the reaction of the pyridyl and quinolyl aldehydes with 3-aminopropyltriethoxysilane. Model ligands were characterized by FT-IR and 1H NMR spectroscopy while 13C{1H} NMR spectroscopy was additionally used for functional ligand characterization. Functionalized complexes of both Pd(II) and Cu(I) were found to be more thermally stable than their model counterparts. Overall the model Pd(II) complexes showed a higher thermal stability than the model Cu(I) complexes. Ligands (L1-L6) were reacted with either Pd(II) or Cu(I) metal precursors to produce both the model and functionalized Pd(II) (C1-C6) and Cu(I) (C7-C12) metal complexes. These metal complexes were all characterized by FT-IR spectroscopy, 1H NMR and UV/Vis spectroscopy for the model Cu(I) complexes. Functionalized complexes were additionally characterized with 13C{1H} NMR spectroscopy. Siloxane functionalized complexes of Pd(II) and Cu(I) were immobilized on MCM-41 and SBA-15 silica materials to produce heterogenized immobilized catalysts. These immobilized catalysts were characterized by a wide range of solid state techniques including: BET nitrogen adsorption/desorption, scanning electron microscopy (SEM), thermal gravimetric analysis (TGA), ICP-AES, FT-IR spectroscopy, powder XRD and solid state 13C{1H} NMR spectroscopy. ICP-AES and BET surface analysis showed that better complex immobilization occurred for SBA-15 supported materials despite SBA-15 having a significantly lower surface area than MCM-41. This higher immobilization was ascribed to the larger pore sizes of SBA-15 (50 Å) vs. that of MCM-41 (26 Å). Immobilized catalysts were tested for the oxidation of benzyl alcohol to benzaldehyde. Immobilization had a positive effect on the catalytic activity of the Pd(II) complexes with higher conversions being observed for immobilized Pd(II) catalysts when compared to their model analogues. Overall the MCM-41 immobilized Pd(II) catalysts showed a higher increase in activity than SBA-15 immobilized catalysts. For Ti-doped supports a generally higher activity was seen for the Ti-SBA-15 system. The Cu(I) systems however were not as effective in the oxidation reactions. / AFRIKAANSE OPSOMMING: In hierdie tesis word die sintese van `n wye reeks model sowel as gefunksioneerde N-(n-propiel)-1-(2-piridiel en kinoliel)-imien ligande (L1-L6) beskryf. Gefunksioneerde ligande (L4-L6) is gevorm deur die reaksie van piridiel en kinoliel aldehied met 3-amniopropieltriëtoksiesilaan. Model ligande is gekaraktariseer deur FT-IR en 1H KMR spektroskopie terwyl 13C{1H} KMR spektroskopie addisioneel gebruik is vir die karaktarisering van die gefunksioneerde ligande. Ligande (L1-L6) is gereageer met Pd(II) of Cu(I) metaal voorgangers om beide model sowel as gefunksioneerde Pd(II) (C1-C6) en Cu(I) (C7-C12) metaal komplekse op te lewer. Hierdie metaal komplekse is almal gekaraktariseer deur FT-IR, 1H KMR en UV/Vis spektroskopie vir die model Cu(I) komplekse. Gefunksionalseerde komplekse is addisioneel gekaraktariseer deur gebruik te maak van 13C{1H} KMR spektroskopie. Dit is gevind dat gefunksionaliseerde komplekse van beide Pd(II) sowel as Cu(I) termies meer stabiel was as hulle ooreenstemmende model komplekse. Oor die algemeen het die Pd(II) komplekse hoër termiese stabiliteit as die Cu(I) komplekse getoon. Siloksaan gefunksioneerde komplekse van Pd(II) en Cu(I) is geimmobiliseer op MCM-41 en SBA-15 silika materiale om heterogene geimmobiliseerde katalisatore op te lewer. Hierdie geimmobiliseerde katalisatore is gekaraktariseer deur van `n wye reeks vaste toestand tegnieke gebruik te maak. Hierdie suit in: SEM, TGA, ICP-AES, FT-IR, poeier XRD en vaste toestand 13C{1H} KMR spektroskopie. ICP-AES en BET oppervlak analieses het getoon dat beter kompleks immobilisering vir die SBA-15 silika material plaas gevind het, ondanks die feit dat SBA-15 `n laer oppervlak area beskik. Hierdie hoër graad van immobilisering is toegeskryf aan die groter poriegrootte van SBA-15 (50 Å) teenoor die van MCM-41 (26 Å). Geimmobiliseerde katalisatore is getoets in die oksidasie van bensielalkohol na bensaldehied. Dit is gevind dat die immobilisering van die Pd(II) komplekse op die silika materiaal `n positiewe uitwerking op die aktiwiteit van die katalitiese van die komplekse gehad het. Die hoogste toename in aktiwiteit is gesien vir geimmobiliseerde Pd(II) katalisatore wanneer hulle met hul ooreenstemmende model komplekse vergelyk is. Oor die algemeen is gevind dat MCM-41 geimmobiliseerde Pd(II) katalisatore n hoër toename in aktiwiteit getoon het as die van SBA-15. Vir die Ti-gedokterde silika materiale het die Ti-SBA-15 sisteem oor die algemeen `n hoër aktiviteit getoon as die Ti-MCM-41 sisteem. Die Cu(I) sisteme was egter nie so effektief in oksidasie reaksies nie.

A range of mesoporous materials based on SBA-15 have been prepared and characterised. The materials were templated by neutral block copolymer P123, and typically have a hexagonal (p6mm) pore structure, with high surface areas and narrow pore size distributions. The removal of the surfactant template by calcination and solvent extraction has been investigated. The aqueous stability of this material, and the hydrolysis of the surface was studied. Organic functional groups were incorporated into the silica surface by co-condensation, or by post synthesis grafting. A range of functional groups were incorporated, including amine, carboxy, allyl and thiol groups. The pore size of the materials was controlled by the addition of trimethoxybenzene during synthesis, which significantly increased the pore size and uptake capacity of the materials. The adsorption of CALB by SBA-15 was investigated, with support materials extracted by calcination or solvent extraction. Rapid uptake at high loading was observed, with a maximum loading of 450 mg g-1 measured. The leaching of the enzyme from the support was investigated, and found to be high with unfunctionalised supports. The leaching from functionalised supports incorporating sulfur groups was significantly reduced. The activity of the immobilised CALB was measured by tributyrin hydrolysis in aqueous media, and by enantioselective transesterification of (R)-1-phenylethanol in organic media. The effect of surface functionalisation for reusability and thermal stability in aqueous systems was investigated. Preliminary studies of supported CALB for dynamic kinetic resolution were carried out, with an investigation of acidic zeolites and a mesoporous supported catalyst for 1-phenylethanol racemisation. The encapsulation of immobilised CALB was investigated, and the activity and reusability of these systems studied.

N-acylation of phosphatidylethanolamine (PE) with free fatty acids catalyzed by N-acyl phosphatidylethanolamine (NAPE) synthase was reported in cotyledons of 24-h-old cotton seedlings. Here I report subcellular localization of this enzyme. Differential centrifugation, sucrose density gradient fractionation,aqueous two-phase partitioning and electron microscopy techniques were utilized to elucidate subcellular site(s) of NAPE synthase. Marker enzymes were used to locate organelles in subcellular fractions. Differential centrifugation indicated that NAPE synthase is present in more than one organelle and it is a membrane bound enzyme. Sucrose density gradient fractionations indicated that NAPE synthase is present in membranes derived from endoplasmic reticulum (ER),Golgi and possibly plasma membrane (PM) but not mitochondria, glyoxysomes or plastids. Aqueous two-phase partitioning experiments with cotton and spinach tissues supported these results but Goigi appeared to be the major site of NAPE synthesis. Electron microscopy of subcellular fractions was used to examine isolated fractions to provide visual confirmation of our biochemical results. Collectively, these results indicate that NAPE is synthesized in plant ER, Golgi and possibly PM.

O presente trabalho teve como objetivo o aproveitamento da matéria-prima residual óleo fúsel como fonte de baixo custo de álcool isoamílico para a síntese de ésteres com diferentes propriedades pela rota enzimática. Utilizou-se como proposta a esterificação do óleo fúsel com ácidos carboxílicos de diferentes tamanhos de cadeia carbônica mediada pela enzima lipase em processos descontínuo ou contínuo, visando obter produtos com propriedades aromáticas, emulsificantes e lubrificantes. A lipase selecionada de Rhizopus oryzae foi posteriormente imobilizada em diferentes suportes sílica-β-ciclodextrina (SiO2- βCD), sílica-hidroxietilcelulose (SiO2-HEC) ou co-polímero de estireno de divinilbenzeno (STY-DVB) e utilizada como biocatalisador para mediar as reações de esterificação do óleo fusel com os ácidos caprílico, láurico e oleico. Entre os suportes testados, apenas a sílica-hidroxietilcelulose (SiO2-HEC) apresentou resultados insatisfatórios em função da elevada afinidade desta matriz em adsorver a água formada como subproduto na reação de esterificação. Para cada par de óleo fúsel e ácido carboxílico, as condições adequadas para obtenção dos produtos alvos foram estabelecidas e os produtos resultantes analisados de acordo com a finalidade proposta. No caso do caprilato de isoamila, as condições reacionais foram determinadas por planejamento experimental, indicando que a formação do éster foi influenciada somente pela variável razão molar ao nível de 95% de confiança. O modelo matemático proposto permitiu prever as condições que favorecem o alcance de elevados rendimentos de formação do éster, sendo a reação maximizada (82% em 24 h) para meios reacionais constituídos de óleo fúsel e ácido caprílico na razão molar de 1:1,5 e temperatura de 45 ºC. O perfil sensorial do éster foi confirmado com auxílio do nariz eletrônico. Com relação ao laurato de isoamila foi possível estabelecer o processo em regime continuo utilizando a lipase de Rhizopus oryzae imobilizada em STY-DVB operando o sistema com concentrações elevadas do substrato que forneceu produtividades elevadas (1598,68 ± 135,79 μmol g-1 min-1) do éster com excelente capacidade de dispersão de pigmentos que pode ser utilizado na formulação de cosméticos com rotulagem de produto natural. Finalmente, com relação ao éster oleato de isoamila, foi constatado limitado desempenho da lipase Rhizopus oryzae imobilizada em STY-DVB devido baixa atividade sintética deste biocatalisador para ácidos carboxílicos insaturados. Mesmo assim, foi possível obter produtividades volumétricas da ordem de 286, 20 ± 18,46 μmol g-1 min-1, utilizando uma configuração de reator de leito empacotado em dois estágios acoplado a uma coluna extratora de água. As propriedades do produto formado atenderam as especificações recomendas para uso como biolubrificante. Desta forma, o presente projeto de tese demonstrou a potencialidade da aplicação do óleo fusel como fonte de baixo custo do álcool isoamílico na obtenção ésteres com propriedades adequadas para uso como aroma, emulsificante e lubrificante. O aproveitamento deste resíduo não somente retira um composto indesejado do meio ambiente, mas também permite a geração de diferentes produtos de interesse industrial, constituindo-se, assim, em um forte apelo ambiental. / The aim of the present work was to investigate the feasibility of using fusel oil as a source of low cost feedstock based on isoamyl alcohol for the synthesis of esters with different properties by enzymatic route. For this, it was proposed to perform the esterification reaction of fusel oil with carboxylic acids with different sizes of carbonic chain mediated by the enzyme lipase under batch or continuous runs, in order to obtain products with aromatic, emulsifying and lubricant properties. Rhizopus oryzae lipase immobilized on different supports silica-β-cyclodextrin (SiO2-βCD), silica-hydroxyethylcellulose (SiO2- HEC) or co-polymer of styrene divinylbenzene (STY-DVB) were chosen as biocatalysts to mediate the esterification reactions of fusel oil with caprylic, lauric and oleic acids. Among the supports tested, only the silica hydroxyethylcellulose (SiO2-HEC) provided unsatisfactory results due to its high affinity to adsorb the water generated as byproduct. For each pair of fusel oil and carboxylic acid, appropriate conditions for obtaining the target product were established, and the resulting product further analyzed according to its proposed use. In the case of isoamyl caprylate, reaction conditions were determined by factorial design, indicating that the ester formation was influenced only by the molar ratio at 95% of confidence level. The proposed mathematical model allowed predicting the conditions that favor the achievement of high yields of ester formation, being maximized (82% in 24 h) with substrate consisting of fusel oil and caprylic acid at molar ratio of 1:1.5 and temperature of 45°C. The sensory profile of the ester was confirmed with help of the electronic nose. With respect to isoamyl laurate it was possible to establish a stable process under continuous flow using Rhizopus oryzae lipase immobilized on STY-DVB feeding the reactor with high substrate concentrations, providing high ester productivity (1598.68 ± 135.79 g-1 μmol min-1) with excellent dispensability of pigments that can be used in cosmetic formulation label as natural product. Finally, with regards to the isoamyl oleate, the performance of Rhizopus oryzae lipase immobilized on STY-DVB was limited due to its low activity towards unsaturated carboxylic acids. Still, it was possible to obtain volumetric productivity of about 286. 20 ± 18.46 μmol g-1 min-1 using two-stage packed bed reactor coupled with a water column extraction. The properties of the formed product meet the specifications recommend for use as biolubricant. In this way, this work demonstrated the feasibility of applying the fusel oil as a source of low-cost isoamyl alcohol to obtain esters with appropriate properties for use as flavor, emulsifier or lubricant. The use of this waste material not only removes unwanted compounds from the environment, but also allows the generation of different products of industrial interest, thus having a strong environmental appeal.

A descoberta de inibidores seletivos é extremamente importante para o desenvolvimento de novos fármacos que possam ser usados no tratamento de pacientes diagnosticados com a doença de Alzheimer (DA). Neste contexto, o desenvolvimento de métodos de triagem para a identificação de novos compostos biologicamente ativos se torna interessante. Butirilcolinesterase (BChE, EC 3.1.1.8) é uma serina hidroxilase que está classicamente associada à hidrólise do neurotransmissor acetilcolina (ACh) formando colina e ácido acético. Este trabalho descreve a imobilização covalente da BChE de soro humano nas paredes internas de capilares de sílica fundida utilizando o agente espaçador glutaraldeído, e sua aplicação na triagem de inibidores seletivos. O ICER-BChE resultante foi conectado a um sistema de cromatografia líquida de alta eficiência com monitoramento on line da atividade catalítica, envolvendo detecção UV. Após os estudos das melhores condições cromatográficas, variações de pH e vazão e a influência de solventes orgânicos na atividade enzimática o método foi validado com o uso de inibidores padrões. O maior valor obtido com o ICER-BChE no parâmetro cinético, constante de Michaelis KM = 33,6 ± 6,9 mM, comparado com a enzima em solução, KM = 0,12 ± 0,02 mM, evidencia o efeito da imobilização sobre a afinidade pelo substrato. No entanto, houve retenção da atividade catalítica e seletividade frente a inibidores padrão. O método foi aplicado na triagem de novos ligantes utilizando cinco coleções de diferentes classes de compostos, entre derivados cumarínicos, complexos metálicos com cobre (Cu), complexos metálicos com cobalto (Co) e zinco (Zn), glicosídeos, e derivados de fenilpropanóides e ácido barbitúrico. Desta triagem foram selecionados sete compostos promissores com os quais foram realizados os estudos sobre a potência mínima inibitória (IC50) e destes quatro foram escolhidos para estudos de mecanismos de ação utilizando o ICER-BChE. Dos complexos metálicos os melhores compostos foram o HPTBCu (IC50 = 8,74 ± 1,5 µM, Ki = 9,6 ± 0,5 M), o NarBCu (IC50 = 8,0 ± 1,4 µM, Ki = 2,0 ± 0,1 M) ambos com mecanismo competitivo o HesFCu (IC50 = 13,6 ± 2,9 µM), o NNINABCu (IC50 = 94,8 ± 16) e o NarFCu (IC50 = 81,7 ± 13). Dos derivados cumarínicos os compostos 17 (IC50 =109 ± 21 µM, Ki = 108 ± 10 M) e 19 (IC50 =128 ± 28 µM, Ki =36.0 ± 5.0 M) apresentaram mecanismo incompetitivo. Os resultados demonstraram que a abordagem proposta é útil na triagem on line de inibidores seletivos, pois fornece resultados rápidos, precisos e reprodutíveis. / The discovery of selective inhibitors is extremely important for the development of drugs that can be used in the treatment of patients diagnosed with the Alzheimer disease (AD). In this context, the development of screening methods for the identification of new, biologically active compounds is a challenging task. Butyrylcholinesterase (BChE, EC 3.1.1.8) is a serine hydroxylase that is classically associated with the hydrolysis of the neurotransmitter acetylcholine (ACh), which yields choline and acetic acid. This paper describes the development of capillary enzyme reactors (ICERs) containing BChE from the human serum, covalently immobilized onto silica fused capillaries, using glutaraldehyde as spacer, and its application in the screening of selective inhibitors. The resulting BChE-ICER was connected to a liquid chromatography system high efficiency where monitoring of activity was online involving UV detection. After studying the best chromatographic conditions, pH variations, flow-rate and the influence of organic solvents on enzyme activity method was validated using standard inhibitors. The higher value obtained with the BChE-ICER in kinetic parameter, constant Michaelis KM = 33.6 ± 6.9 mM, compared with the enzyme in solution, KM = 0.12 ± 0.02 mM, shows the effect of immobilized on the affinity substrate. However, there was retention of catalytic activity and selectivity towards standard inhibitors. The method was applied in the screening of new ligands using collections of five different compounds, among coumarin derivatives, metal complexes with copper (Cu) metal complexes with cobalt (Co) and zinc (Zn), glycosides, phenylpropanoids and derivatives, and barbituric acid. These screenings were selected seven promising compounds with which the studies were made on the minimum inhibitory potency (IC50) and these four were chosen for studies of mechanisms of action using the ICER-BChE. Of the compounds metal complexes best were HPTBCu (IC50 = 8,74 ± 1,5 µM, Ki = 9,6 ± 0,5 M), NarBCu (IC50 = 8,0 ± 1,4 µM, Ki = 2,0 ± 0,1 M) both competitive mechanism, the HesFCu (IC50 = 13.6 ± 2.9 µM), NNINABCu (IC50 = 94.8 ± 16 µM) and NarFCu (IC50 = 81.7 ± 13 µM). Of coumarin derivatives, 17 (IC50 =109 ± 21 µM, Ki = 108 ± 10 M) and 19 (IC50 =128 ± 28 µM, Ki =36.0 ± 5.0 M) showed mechanism uncompetitive. The results demonstrated that the proposed approach is useful in screening for selective inhibitors online because it provides quick results, accurate and reproducible.

O presente trabalho teve por objetivo avaliar condições de imobilização da levedura Scheffersomyces stipitis NRRL Y-7124 pelo método do aprisionamento em gel de alginato de cálcio visando à produção de bioetanol em biorreator STR tipo cesta à partir de hidrolisado hemicelulósico de bagaço de cana-de-açúcar. Primeiramente, realizou-se as etapas de obtenção, destoxificação e caracterização do hidrolisado hemicelulósico de bagaço de cana-de-açúcar. Realizou-se em seguida um screening objetivando a seleção de um meio de cultivo adequado para a produção de etanol por esta levedura. O meio escolhido foi aquele onde se suplementou o hidrolisado com extrato de levedura (3,0 g/L), peptona (5,0 g/L), (NH4)2SO4 (2,0 g/L) e CaCl2 (0,1 g/L), onde verificou-se um fator de conversão de xilose à etanol (Yp/s) de 0,33 g/g. As condições de imobilização da levedura foram então avaliadas por planejamento fatorial 23 completo onde os fatores concentração de alginato de sódio, concentração do cloreto de cálcio e tempo de cura foram investigados. Após a análise estatística, as condições 2% de alginato de sódio, 0,1M de cloreto de cálcio e tempo de cura de 12 horas foram fixadas para as etapas seguintes. Nestas condições, avaliou-se então a influência da concentração de células à serem imobilizadas e agitação durante a fermentação a partir de um planejamento fatorial 22 completo, definindo-se assim 10 g/L de células e 100 rpm como condições ideais. Após a determinação das condições de imobilização do processo, verificou-se a estabilidade das células imobilizadas em repetidos ciclos fermentativos, para isso cinco bateladas repetidas em frascos Erlenmeyer foram realizadas. Observou-se que apesar da levedura assimilar xilose e produzir etanol em todos os ensaios, uma diminuição na eficiência da fermentação foi verificada, diminuindo em 24% da terceira para a quarta batelada, indicando assim que a levedura imobilizada era viável para o sistema de batelada repetida em até 3 ciclos nas condições estudadas. Iniciou-se então ensaios fermentativos em biorreator STR tipo cesta, realizando-se ensaios em meio sintético e em hidrolisado hemicelulósico. Observou-se reprodutibilidade nos ensaios utilizando os diferentes meios, com um valor de Yp/s de 0,21g/g e uma produtividade volumétrica de 0,15 g/L.h em ambos os ensaios. Fermentações em sistema de bateladas repetidas foram realizadas neste biorreator STR tipo cesta. Realizou-se cinco ciclos consecutivos, ao final dos quais observou-se comportamento semelhante às bateladas repetidas realizadas em frascos Erlenmeyer, na qual a partir de três ciclos a capacidade fermentativa da levedura S. stipitis diminuiu, apresentando uma produtividade volumétrica em torno de 0,16 g/L.h nas três primeiras bateladas. O gel de alginato de cálcio apresentou considerável estabilidade em sistema de bateladas repetidas indicando a possibilidade de sua utilização nesse processo. Embora os resultados obtidos neste trabalho sejam inferiores aos observados com células livres por outros autores, os mesmos demonstraram o potencial do emprego do gel de alginato de cálcio e da levedura Scheffersomyces stipitis imobilizada para a produção de bioetanol a partir de bagaço de cana-de-açúcar e contribuíram para os conhecimentos sobre a fermentação de hidrolisado hemicelulósico à etanol. / This study aimed to evaluate immobilization conditions for the yeast Scheffersomyces stipitis NRRL Y-7124 entrapped in calcium alginate gel in basket type of STR bioreactor for ethanol production from sugarcane bagasse hemicellulosic hydrolysate. For this purpose, first the steps to obtain the hydrolysate by dilute acid pretreatment, detoxification and characterization of hydrolysate was performed. Then, a screening aiming the selection of a suitable culture medium suitable for ethanol production by this yeast was carried out. The medium which showed maximum ethanol production (Yp/s, 0.33 g/g) was selected to continue the further studies. It was composed by the hydrolyzate supplemented with yeast extract (3.0 g/L), peptone (5.0 g/L), (NH4)2SO4 (2.0 g/L), CaCl2 (0.1g/L). The immobilization conditions of the yeast were then evaluated through a 23 factorial design where the three process variables i.e. concentration of sodium alginate, concentration of calcium chloride and reaction time were investigated. After statistical analysis, the optimum set of conditions (2% of sodium alginate, 0.1 M of calcium chloride and a reaction time of 12 hrs) were set to perform the following steps of this study. Subsequently, the influence of the cell concentration for immobilization and agitation during fermentation were studied considering a factorial design 22. This study revealed that 10 g/L of cells and 100 rpm were the optimum conditions for ethanol production via immobilized systems. After determination of the conditions for immobilization procedure, the stability of the immobilized cells were evaluated by repeated fermentation cycles, for that five repeated batches were performed in Erlenmeyer flasks. It was observed that despite the yeast assimilates xylose and produces ethanol in all assays, a decrease in the efficiency of the fermentation was verified from the third batch, revealing the 65% efficiency in the second batch and 39% in the fourth batch. This behavior indicates that the immobilized yeast is viable for repeated batch system only up to 3 cycles under the employed conditions. Fermentation tests in basket type STR bioreactor were carried out using synthetic medium and hemicellulosic hydrolysate as carbon source. Reproducibility was observed in assays using the different medium with ethanol yield (Yp/s) of 0.21 g/g and a volumetric productivity of 0.15 g/L.h in both assays. Fermentation assay in repeated batch system were carried out in STR basket type bioreactor. Five consecutive fermentation cycles were performed which eventually showed the similar behavior with the repeated batches conducted in Erlenmeyer flasks. The fermentative efficiency of the yeast S. stipitis was considerably good up to three cycles with a volumetric productivity of 0.16 g/L.h followed by a concomitant down fall. The calcium alginate gel showed a considerable stability in the experiments, indicating the viability of its application in repeated batch system. Although the results of this work are inferior to that observed by other authors using free cells, the calcium alginate gel potential is evident and the yeast Scheffersomyces stipitis showed to be capable to produce ethanol in immobilized form, contributing with knowledge for second generation ethanol production from sugarcane bagasse hemicellulosic hydrolysate adopting biochemical platform.

Este trabalho de doutorado apresenta os resultados da avaliação de duas configurações diferentes de reatores com biomassa imobilizada em matrizes cúbicas de poliuretano, denominados reator misto radial de leito fixo (RMRLF), utilizado para o tratamento de esgoto sanitário peneirado e reator aeróbio-anaeróbio horizontal de leito fixo (RAAHLF) para o pós-tratamento de efluente de reator de manta de lodo tipo upflow anaerobic sludge blanket (UASB) tratando esgoto sanitário. O estudo avalia duas propostas de tratamento para remoção biológica de matéria orgânica e nitrogênio por meio do acompanhamento do desempenho dos reatores sob diferentes condições operacionais, quais sejam: variação na carga orgânica aplicada, diferentes dosagens de solução alcalina e variação na taxa de aplicação de ar comprimido para o fornecimento de oxigênio dissolvido aos reatores na etapa de nitrificação. Foram desenvolvidos ensaios para obtenção de dados sobre o comportamento hidrodinâmico no RMRLF, dados sobre transferência e consumo de oxigênio dissolvido e viabilidade da utilização de biogás como doador de elétrons para a desnitrificação autótrofa. Os principais parâmetros de desempenho foram avaliados através de análises físico-químicas das amostras e a observação dos microrganismos envolvidos no processo. O RMRLF operou durante todo o período dos experimentos com a temperatura do líquido no seu interior variando entre o valor mínimo de 14 'GRAUS' Celsius até máximo de 30 'GRAUS' Celsius. Os valores da demanda química de oxigênio (DQO) no efluente do reator mantiveram média inferior a 50 mg/L. A remoção de nitrogênio como nitrato aumentou em 75% após o início do fornecimento do 'H IND.2'S' por meio da injeção de biogás. O RAAHLF operou com a temperatura do líquido no seu interior variando entre mínimo de 14 'GRAUS' Celsius e máximo de 26 'GRAUS' Celsius. Os valores de DQO no efluente, em média, foram inferiores a 66 mg/L e a remoção de nitrato foi incrementada em 87% após o fornecimento do 'H IND.2'S' pela injeção de biogás na câmara anóxica do reator. Os resultados demonstram o potencial destes reatores como alternativa para o tratamento e pós-tratamento de esgoto sanitário com resultados promissores de remoção de matéria orgânica e nitrogênio. / This work presents results of the evaluation of two different immobilized biomass reactor configurations, referred as radial flow anaerobic/aerobic immobilized biomass reactor (RAAIB) utilized for the treatment of the sanitary screened wastewater and horizontal aerobic/anaerobic immobilized biomass reactor (HAAIB) for the post-treatment of an upflow anaerobic sludge blanket reactor (UASB) treating sanitary wastewater. This study evaluates the two proposals for the biological organic matter and nitrogen removal by the evaluation of the reactors performance under several operational conditions, such as organic matter load variation, alkalinity solution feeding, variation on the compressed air supply for the nitrification step and biogas supply for the denitrification process. Assays were developed in order to obtain data on the hydrodynamic behavior in the RAAIB reactor, oxygen transfer and consumption and the viability of use biogas as electron donor for the autotrofic denitrification. The main performance parameters were measured thru physical-chemical analysis and microscopic observations. The RAAIB reactor was operated during all the experiment period with its liquid temperature varying from 14 'DEGREES' Celsius to 30 'DEGREES' Celsius. The chemical oxygen demand (COD) values in the effluent presented a mean value below 50 mg/L. The nitrogen as nitrate removal efficiency incresead up to 75% after supplying 'H IND.2'S' thru biogas injection. The HAAIB reactor operated with temperatures varying from 14 'DEGREES' Celsius to 26 'DEGREES' Celsius. The COD values in the effluent presented a concentration value lower than 66 mg/L and the nitrate removal increased 87% after supplying 'H IND.2'S' thru the biogas injection in the anoxic reactor. The collected data proved the viability of the use of these reactors as an alternative for the treatment and post-treatment of sanitary wastewater due to the promising results obtained in the assays performed in the reactors.

Resíduos de formaldeído são descarregados de diversas formas no ambiente, resultantes de muitos processos industriais, e de seu uso como conservantes. A pesquisa por tecnologias adequadas para o tratamento de formaldeído aponta processos físico-químicos e biológicos, com atenção especial para os processos anaeróbios por constituírem sistemas compactos e de baixo consumo energético. No entanto, algumas lacunas presentes na pesquisa sobre tratamento anaeróbio de formaldeído e alguns pontos de discordância devem ser esclarecidos. Em muitos casos, esses resíduos são descartados de forma intermitente, motivando a opção por processos em batelada. Nesse contexto, o presente estudo avaliou a degradação de formaldeído em reator anaeróbio operado em bateladas seqüenciais, contendo biomassa imobilizada em espuma de poliuretano. O desempenho do reator foi monitorado para várias concentrações afluentes de formaldeído, variando de 31,6 a 1104,4 mg/L. Os resultados obtidos indicaram excelente estabilidade do reator e eficiência de remoção de formaldeído acima de 99%. Entretanto foi constatado acúmulo de matéria orgânica no efluente devido à presença de ácidos orgânicos, principalmente acético e propiônico. Essa constatação levanta um questionamento importante sobre a rota anaeróbia de degradação do formaldeído, que pode diferir substancialmente do que foi registrado na literatura. Os ácidos gerados não foram degradados pela biomassa exposta ao formaldeído, contudo poderiam ser facilmente removidos com a utilização de um reator em série com inoculo adaptado à remoção de ácidos orgânicos. / Formaldehyde residues are discharged into the environment in several different ways, as a result of several industrial processes, as well as its use as a preservative. Research for suitable technologies for treatment of formaldehyde points to physical-chemical and biological processes, with special attention to anaerobic processes, once they represent compact systems with low energy requirements. Nevertheless, some blank spaces still remaining in the research regarding anaerobic treatment of formaldehyde must be filled, and some points of disagreement must be clarified. In several cases, formaldehyde residues are discharged intermittently, favoring the choice of batch processes. In this context, the present study evaluated the degradation of formaldehyde in an anaerobic sequencing batch reactor, containing biomass immobilized in polyurethane foam matrices. Reactor performance was monitored for different influent formaldehyde concentrations, ranging from 31.6 to 1104.4 mg/L. Results obtained indicate excellent reactor stability and efficiency in formaldehyde removal above 99%. However, accumulation of organic matter was observed in the effluent, due to presence of non-degraded organic acids, especially acetic and propionic. This observation poses an important question regarding the anaerobic route of formaldehyde degradation, which might differ substantially from that reported in literature. The degradation of the generated products could occur in another reactor in serie containing biomass adapted to organic acids removal.

A proposta desta pesquisa foi avaliar a utilização de um sistema combinado de filtro anaeróbio (FAN) com filtro aeróbio (FAE) submerso ligados em série, ambos usando espuma de poliuretano como meio suporte e com fluxo ascendente, como alternativa para remoção matéria orgânica (DQO) e compostos nitrogenados em efluentes de refinarias de petróleo, além da redução da toxicidade do efluente ao micro crustáceo Mysidopsis juniae. O experimento durou 324 dias e foi divido em duas etapas. Inicialmente, o sistema foi operado por 129 dias sem recirculação entre os filtros e nesta etapa houve acréscimo de 12% nas concentrações de amônia no FAN e remoção de 80% no FAE. Quando se usou bicarbonato de sódio para alcalinizar o afluente ao sistema para manter o pH no FAE na neutralidade e alcalinidade acima de 107 mg\'CA\'CO IND.3\'/L, pôde-se alcançar remoções de amônia próxima a 100%. Quanto à remoção de DQO, o filtro anaeróbio removeu 40% e o aeróbio 65%. Em síntese, na primeira etapa o sistema removeu 80% de DQO, 89% de amônia e 35% de nitrogênio. A segunda etapa durou 195 dias e houve recirculação do efluente do filtro aeróbio para o filtro anaeróbio. Nesta etapa, o pH foi mantido na neutralidade e a recirculação foi feita de duas formas. A primeira com recirculação direta entre os filtros a uma razão (R) de reciclo de 2,0 resultou em 43% de remoção de amônia no FAN e 97% no FAE e com esta forma de reciclo houve 62,6% de remoção de nitrogênio total no sistema. A segunda forma de recirculação foi direcionar parte do efluente do filtro aeróbio ao reservatório do afluente ao sistema com R igual a 1,0 resultando em produção de amônia no FAN em 10% e remoção no FAE de, aproximadamente, 100%. Independentemente da forma de recirculação a remoção de DQO foi de 67% e 56% no filtro anaeróbio e aeróbio, respectivamente. A remoção de nitratos no FAN foi de 93% e foi independente da forma de reciclo. Houve 42,5% de remoção de nitrogênio no sistema. Na segunda etapa obtiveram-se as melhores remoções de DQO, amônia e nitrogênio, porém, tanto o afluente quanto o efluente final apresentou CL50 de 0,83% ao microcrustáceo M. juniae, assim, ambas as formas do efluente estão em desacordo com a resolução CONAMA 357/05. Os gêneros Aeromonas, Serratia e Pseudomonas foram dominantes no FAE ao fim da pesquisa. / The purpose of this study was to evaluate the use of a combined system comprised of an anaerobic filter (ANF) and a submerged aerobic filter (SAF) connected in series, both using polyurethane foam as support media and operated in a upflow mode, as an alternative for removing organic matter (COD) and nitrogen compounds in effluents from oil refineries, as well as reducing effluent toxicity to micro crustacean Mysidopsis juniae. The experiment lasted 324 days and was divided into two stages. Initially, the system was operated for 129 days without recirculation between the filters. During this stage, there was an increase of 12% on the ammonia content in the ANF followed by a removal of 80% in the SAF. When sodium bicarbonate was used to alkalize the influent to maintain the pH in the ANF close to neutral and alkalinity above 107 mg\'CA\'CO IND.3\'/L, the removal efficiency of ammonia reached values close to 100%. Regarding to COD removal, the ANF removed 40% and SAF removed 65%. In summary, in the first stage of the research the system removed 80% of COD, 89% of ammonia and 35% of nitrogen. The second stage lasted 195 days and there was a recirculation line from the SAF to the ANF. In this step, the pH was maintained close to neutral and recirculation was done in two different ways. In the first, the recirculation flow was conducted from the SAF direct to the ANF at a rate of 2.0, which resulted in 43% removal of ammonia in the ANF and 97% in the SAF. Moreover, with this form of recycling 62.6% of the total nitrogen was removed from the system. The second way was to direct part of the effluent of the SAF to the tank that contained the influent of the ANF. In this case, the recirculation rate was 1.0, and resulted in a production of 10% of ammonia in the ANF and removal of approximately 100% in the SAF. Independently of the way used for recirculation, the COD removal efficiencies was 67% and 56% in ANF and SAF, respectively. The removal of nitrate in the ANF was 93% and was independent of the form of recycling. There was a 42.5% removal of nitrogen in the system. In the second stage occurred the best removals of COD, ammonia and nitrogen. However, both the affluent and the effluent had LC50 of 0.83% to microcrustacean M. juniae. Therfore, both forms of the effluent are in disagreement with the Brazilian law Resolução CONAMA 357/05. The Aeromonas, Serratia and Pseudomonas were dominant in the SAF at the end of research.