O ácido clavulânico (AC) é um potente inibidor de β-lactamases, sendo utilizado em associação com antibióticos β-lactâmicos. Atualmente, a purificação industrial do AC envolve, principalmente, processos de extração líquido-líquido com solventes orgânicos e etapas cromatográficas. Assim, métodos alternativos como os sistemas micelares de duas fases aquosas (SMDFA), os quais oferecem seletividade na partição de biomoléculas de acordo com sua hidrofobicidade, são de grande interesse. O presente trabalho teve como objetivo estudar a partição do AC em sistemas micelares não iônicos de duas fases aquosas, puros e com adição do sal (NH4)2SO4 ou do polímero sulfato de dextrana (Dx-S). Os estudos de estabilidade do AC mostraram que o fármaco é mais estável em pH 6,5 e temperaturas mais baixas (5 - 20 ºC). Em relação à presença dos aditivos, foi verificado que a adição do Dx-S acarretou em menor perda da estabilidade do AC quando comparado ao (NH4)2SO4, com valor residual ≥ 90% a 35 °C. Na presença dos tensoativos Triton X-114 e Triton X-100, o AC apresentou-se estável, com valor residual de aproximadamente 100%. De acordo com os ensaios de partição, o AC foi recuperado preferencialmente na fase pobre em micelas, tanto nos sistemas TX/tampão quanto TX/sal para ambos os tensoativos, com valores de coeficiente de partição (KAC) ~ 0,7 e rendimento na fase diluída (Yclavd) ~ 75%. A adição do polímero em maiores concentrações (≥ 8% p/p) proporcionou um pequeno aumento nos valores de KAC, porém com valores ainda próximos a 1 - 1,5. Portanto, os resultados demonstraram que a presença dos aditivos não influenciou suficientemente a partição do AC para a fase micelar e, desta maneira, os sistemas TX/tampão mostraram ser mais eficientes para a recuperação do ácido clavulânico na fase pobre em micelas, podendo ser empregados como etapa prévia de extração em um processo biotecnológico. / Clavulanic acid (CA) corresponds to a potent β-lactamase inhibitor that is used in association with β-lactamic antibiotics. The industrial purification of CA usually involves liquid-liquid extraction processes employing organic solvents followed by several chromatographic steps. Therefore, new purification alternatives such as aqueous two-phase micellar systems (ATPS) are of great interest. These systems can provide selectivity in biomolecule partitioning according to hydrophobicity and other molecular properties. Within this context, the main goal of this study was to investigate CA partitioning in aqueous two-phase micellar (nonionic) systems, with and without the addition of (NH4)2SO4 or dextrane sulfate (Dx-S). Stability studies performed with CA indicated that the drug is more stable at pH 6.5 and lower temperatures (5 - 20 ºC). In addition, it was demonstrated that Dx-S addition led to a lower loss of CA stability in comparisson to (NH4)2SO4, with residual values ≥ 90% at 35 °C. The drug was found to be very stable in the presence of the surfactants Triton X-114 and Triton X-100, with residual values around 100%. Regarding CA partitioning in the ATPMS, the drug partitioned preferentially to the micelle-poor phase, irrespective of the surfactante employed and of the presence of (NH4)2SO4,with partition coefficient (KAC) ~ 0.7 and yield in the poor phase (Yclavd) ~ 75%. Nonetheless, the addition of Dx-S in concentrations (≥ 8.0% p/p) resulted in a discrete increase in KAC, with values around 1 - 1.5. Therefore, the results obtained in this work demostrated that the addition of (NH4)2SO4 or Dx-S to ATPMS did not significantly influenced CA partitioning to the micelle-rich phase and, in this context, the systems investigated could be considered more eficiente for CA recovery in the micelle-poor phase, as a previous extraction step of a biotechnological process.

As lipases (EC 3.1.1.3) hidrolisam ligações éster de triacilgriceróis numa interface água-óleo. Por possuírem diversas aplicações industriais, são as enzimas mais utilizadas em sínteses orgânicas e mais de 20% de biotransformações são realizadas com lipases. Os objetivos deste trabalho foram avaliar as condições nutricionais de meios de cultivo (fontes de óleos vegetais e animais, nitrogênio e carbono) para melhorar a produção de lipase por Candida viswanathii além de purificar a enzima em sistema aquoso bifásico e realizar a caracterização parcial da lipase. Entre os meios de cultivos analisados o meio de Vogel proporcionou a maior produção de lipase após 36 horas de cultivo (11,73 U/mL). Após analisar as fontes de carbono lipídicas e não-lipídicas e fontes de nitrogênio, um planejamento experimental de misturas foi aplicado para analisar o efeito de glicerol, lactose e sorbitol sobre a produção de lipase. Os resultados obtidos mostraram que a maior produção de lipase (20,41 U/mL) foi observada com 0,5% de lactose (p/v) e 0,5% (p/v) de sorbitol, sendo observado um aumento de 74% da produção de enzima. Nesse estudo observou-se que a lactose e sorbitol foram utilizados como adjuntos para a produção de lipase. A lipase produzida nas condições anteriores foi purificada em sistema aquoso bifásico (SAB) formado por polietilenoglicol (PEG) e fosfato de potássio. O maior coeficiente de partição (1,34) foi encontrado no ensaio com PEG 4000/fosfato a Temperatura de 40 °C e pH 7,0, bem como o maior balanço de atividade (50,73%). Na caracterização parcial, a lipase não sofreu influência da temperaturana faixa de 20 – 60 °C e obteve atividade máxima em pH 8,1. A enzima apresentou também elevada estabilidade em solventes orgânicos como metanol e etanol, sendo estas propriedades consideradas importantes para aplicações em processos biotecnológicos. / Lipases (EC 3.1.1.3) hydrolyze ester bonds of triacylglycerols at a water-oil interface. By they have several industrial applications, they are the enzymes most used in organic synthesis and more than 20% of biotransformations are performed with lipases. The objectives of this work were to evaluate the nutritional conditions of culture media (plant and animal oils, nitrogen and carbon) to improve the production of lipase by Candida viswanathii in addition to purifying the enzyme in a biphasic aqueous system and to realize the partial characterization of lipase. Among the culture media analyzed, the Vogel medium provided the highest lipase production after 36 hours of culture (11,73 U/mL). After analyzing the lipid and non-lipid carbon sources and nitrogen sources, an experimental design of blends was applied to analyze the effect of glycerol, lactose and sorbitol on lipase production. The results showed that the highest production of lipase (20,41 U/mL) was observed with 0.5% lactose (w/v) and 0.5% (w/v) sorbitol, with an increase of 74% of enzyme production. In this study it was observed that lactose and sorbitol were used as adjuncts for the production of lipase. The lipase produced under the above conditions was purified in a aqueous two-phase systems (ATPS) consisting of polyethylene glycol (PEG) and potassium phosphate. The highest partition coefficient (1,34) was found in the PEG 4000/phosphate assay at temperature of 40 °C and pH 7,0, as well as the highest activity balance (50,73%). In the partial characterization, the lipase was not influenced by the temperature range of 20 - 60 °C and obtained maximum activity at pH 8,1. The enzyme also showed high stability in organic solvents such as methanol and ethanol, and these properties are considered important for applications in biotechnological processes.

O ácido clavulânico (AC) é um potente inibidor de β-lactamases utilizado na área médica. Métodos alternativos, econômicos e simples para sua purificação são de grande interesse. Este trabalho objetivou produzir e extrair AC de Streptomyces spp. por fermentação extrativa utilizando sistema de duas fases aquosas (SDFA) - polietileno glicol (PEG)/sais fosfato. Foi selecionado o melhor produtor de AC entre sete linhagens de Streptomyces spp. Avaliou-se a influência de cinco fatores no cultivo do melhor produtor em frascos agitados (pH, temperatura, velocidade de agitação, concentrações das fontes de nitrogênio e de carbono), utilizando planejamento experimental estatístico. Definidas as melhores condições de cultivo, foram estudadas a produção e a extração do AC em fermentação extrativa utilizando SDFA em frascos agitados e em sistema descontínuo utilizando biorreator. Em biorreator também foram realizados o estudo termodinâmico do processo de fermentação nas condições ótimas obtidas nas etapas anteriores e a determinação do coeficiente volumétrico de transferência de massa (kLa), comparando os sistemas de fermentação no meio de cultivo simples (SF) e fermentação extrativa utilizando sistema SDFA PEG/sais fosfato (SFE) sem e com crescimento microbiano. A linhagem de Streptomyces selecionada como a melhor produtora de AC foi a DAUFPE 3060, a qual apresentou a maior produção desse inibidor, 494 mg/L em 48h, em frascos agitados nas condições: pH 6,0, 32°C, 150 rpm, 5 g/L de glicerol e 20 g/L de farinha de soja. Após a etapa de otimização realizada para o estudo da temperatura e da concentração de farinha de soja, variáveis mais significativas no estudo de seleção, a temperatura e a concentração de farinha de soja ótimas, foram 32°C e 40 g/L, respectivamente, com produção de 629 mg/L de AC em 48h. O estudo termodinâmico confirmou que a temperatura de 32°C é a máxima de produção do AC; após esse valor, inicia-se, gradualmente, a degradação do AC. No estudo da determinação do coeficiente de transferência de massa, kLa, sem crescimento microbiano, observaram-se valores maiores de kLa para o SF, devido à viscosidade do PEG utilizado no SFE. A massa molar do PEG e a velocidade de agitação foram as variáveis que mais influenciaram na extração de AC no SFE em frascos agitados, apresentando comportamento semelhante em biorreator. E, finalmente, o estudo da transferência de oxigênio do SFE utilizando SDFA com crescimento microbiano foi avaliado para otimizar a produção e a extração de AC. Os resultados obtidos demonstraram que existe uma faixa ideal de velocidade de agitação e de aeração para evitar o rompimento celular e aumentar a recuperação de AC. / Clavulanic acid (CA) is a potent inhibitor of β-lactamases used in the medical field. Alternative methods, economic and simple purification are of great interest. This PhD project aims to produce and extract clavulanic acid of Streptomyces spp. By extractive fermentation using aqueous two-phase system (ATPS) - Polyethylene glycol (PEG)/phosphate salts. The best producer of clavulanic acid among seven strains of Streptomyces spp was selected. The influence of five factors in the cultivation of the best producer in flasks (pH, temperature, agitation velocity, concentrations of nitrogen and carbon sources) using statistical experimental design was evaluated. Defined the best cultivation conditions, the production and extraction of clavulanic acid by extractive fermentation using ATPS in flasks and in a batch system using a bioreactor was analyzed. In batch system using a bioreactor were also carried out the thermodynamic study of the fermentation process in optimum conditions determined in previous steps and also determined the volumetric mass transfer coefficient (kLa) comparing the fermentation systems in simple culture medium (SF) and in a extractive fermentation using aqueous two-phase system (ATPS) PEG/phosphate salts (SEF) medium with and without microbial growth. A strain of Streptomyces spp. selected as the best producer of AC was DAUFPE 3060, which showed the highest production of this inhibitor, 494 mg/L at 48h, in flasks under the conditions of pH 6.0, 32 °C, 150 rpm, 5 g/L of glycerol and 20 g/L of soybean flour. After the optimization step, the most significant variables in the study selection, temperature and concentration of soybean flour, were studied. The optimal values were 32 °C and 40 g/L of temperature and soybean flour concentration, respectively, with production of 629 mg/L of CA after 48h of cultivation. The thermodynamic study confirmed that 32 °C is the maximum temperature production of CA, after this value, starts gradually, the degradation of CA. In the study of volumetric mass transfer coefficient, kLa, without microbial growth, showed higher values of kLa for the SF, because the high viscosity of the PEG used in the SFE. The PEG molar mas and agitation velocity were the variables that most influenced the extraction of CA in flasks using a SFE, with similar behavior in a bioreactor. Finally, the study of oxygen transfer rate in SFE using ATPS with microbial growth was evaluated to optimize the production and extraction of CA. The results showed that there is an ideal range of agitation and aeration to prevent cell disruption and increase the CA recovery.

Este trabalho tem como finalidade obter condições de recuperar e purificar a pró-toxina épsilon e uma protease produzida pelo Clostridium perfringens através do uso da técnica de extração líquido-líquido em sistema de duas fases aquosas (SDFA) para utilização na produção de vacinas. A aplicação do sistema de duas fases aquosas é proposta como alternativa para a purificação, pois permite a separação e análise de partículas biológicas. Esta técnica é aconselhável para purificação em larga escala pela possibilidade de partição seletiva com altos rendimentos, além de apresentar uma boa relação custo-benefício. Foram construídas as curvas binodais que foram utilizadas para análise da composição dos sistemas de duas fases aquosas formados por polietileno glicol e citrato de sódio, como também a extração e recuperação da pró-toxina épsilon e da protease produzidas por Clostridium perfringens. As curvas binodais foram construídas utilizando PEG 400, 550, 1000, 1500, 3350 e 8000 em diferentes valores de pH (6,0; 6,5; 7,0; 7,5 e 8,0) e água para formação do sistema. Também foram construídas curvas na presença de caldo clarificado em substituição à água. Foi avaliada ainda a estabilidade da pró-toxina épsilon, antes da extração, e da protease antes e após a extração, frente às variações de pH e temperatura. Um planejamento experimental ('2pot.8-3') foi usado para avaliar a influência das variáveis concentração e massa molar do PEG, concentração de citrato, pH, concentração de NaCl, fator de diluição do extrato, temperatura e massa total do sistema na extração da pró-toxina épsilon. A partição de uma protease presente no meio fermentado de C. perfringens foi estudada através do uso de três planejamentos experimentais completos (dois do tipo '2pot.4' e um do tipo '2pot.3') que avaliaram a influência das variáveis concentração e massa molar do PEG, concentração de citrato e pH. Três variáveis-resposta foram obtidas (aumento de pureza, coeficiente de partição e recuperação da enzima). Os resultados atingidos foram: coeficiente de partição de 0,57, aumento de pureza de 4,2 com uma recuperação de 131% da atividade enzimática na fase superior do sistema. O sistema de extração que proporcionou as melhores condições de extração foi constituído por: PEG 10000 (g/mol) e concentração de 22% (m/m), concentração de citrato de 8% (m/m) e pH 8,5. A protease permaneceu estável (durante 48 h), mesmo após a extração, nas temperaturas de 5°C e 25°C e nos valores de pH de 6,0 a 9,0. / The purpose of this work is to obtain best conditions of recovery and purification of proteins (epsilon prototoxin and a protease) produced by Clostridium perfringens through the use of the liquid-liquid extraction by aqueous two-phases systems (ATPS). The application of these systems is proposed as alternative for protein purification, because it allows the separation and analysis of biological particles. This technique is advisable process purification applied to large scale since it provides a selective partition with high yields, and good cost-benefit ratio. The binodal curves were constructed and used to determine the system composition based on polyethylene glicol and citrate concentrations. The binodal curves were built by using PEG 400, 550, 1000, 1500, 3350 and 8000 g/mol at different pH values (6.0; 6.5; 7.0; 7.5 and 8.0). The curves were, initially, built in the presence of water and, later, with clarified fermented broth. The differences in the curve profiles (water versus broth) helped to explain the phase separation behaviour. The stability, as a function of pH and temperature, of the epsilon prototoxin was evaluated before the extraction, while the stability of the protease was evaluated before and after the extraction. An experimental design ('2pot.8-3') was used to evaluate the influence of the following variables on epsilon prototoxin extraction: concentration and molar mass of PEG, citrate concentration and NaCl concentrations, pH, dilution factor of the extract, temperature and total mass of the system. However, the partition of the protease was studied through the use of the three full different experimental designs (two of the type '2pot.4', and one of the type '2pot.3') that evaluated the influence of the following variables: concentration and molar mass of PEG, citrate concentration and pH. Three parameter responses were obtained: purification factor; coefficient partition; and recovery yield of the enzyme. The results were satisfactory: partition coefficient = 0.57, purification factor = 4.2; yield = 131%. The extraction conditions which provided the best results were: molar mass of PEG 10000 (g/mol); concentration of PEG of 22% (w/w); concentration of citrate of 8% (w/w); and pH 8.5. The protease was stable (during 48h), even after the extraction, in the temperatures of 5°C and 25°C, and at pH values of from 6.0 to 9.0.

The bacterium Escherichia coli (E. coli) is in many situations an ideal host for production of recombinant proteins, since it generally provides a rapid and economical means to achieve sufficiently high product quantities. However, there are several factors that may limit this host’s ability to produce large amounts of heterologous proteins in a soluble and native form. For many applications a high purity of the recombinant protein is demanded, which implies a purification strategy where the product efficiently can be isolated from the complex milieu of host cell contaminants. In this thesis, different strategies based on both rational and combinatorial genetic engineering principles have been investigated, aiming at improving and facilitating recombinant E. coli protein production and purification. One objective was to improve the PEG/salt aqueous two-phase system (ATPS) purification process of the lipase cutinase, by increasing the selectivity of the protein for the system top-phase. Peptide tags, with varying properties, were designed and genetically fused to the C-terminal end of ZZ-cutinase. Greatly increased partitioning values were observed for purified protein variants fused to tryptophan containing peptide tags, particularly a (WP)4 peptide. The partitioning properties of the ZZ-cutinase-(WP)4 protein were also retained when added to the ATPS directly from an E. coli total cell disintegrate, emphasizing the applicability of this genetic engineering strategy for primary protein purification in ATPSs. Further on, a combinatorial library approach using phage display technology was investigated as a tool for identification of peptide tags capable of improving partitioning properties of ZZ-cutinase in an ATPS. Repeated ATPS-based partitioning-selection cycles of a large phagemid (pVIII) peptide library, resulted in isolation of phage particles preferentially decorated with peptides rich in tyrosine and proline residues. Both a peptide corresponding to a phage library derived peptide sequence as well as peptides designed based on information of amino acid appearance frequencies in later selection rounds, were shown to improve partitioning several-fold when genetically fused to the C-terminal end of ZZ-cutinase. From the two- to four–fold increased production yields observed for these fusion proteins compared to ZZ-cutinase-(WP)4, it was concluded that the selection system used allowed for selection of desired peptide properties related to both partitioning and E. coli protein production parameters. Bacterial protein production is affected by several different mRNA and protein sequence-related features. Attempts to address single parameters in this respect are difficult due to the inter-dependence of many features, for example between codon optimization and mRNA secondary structure effects. Two combinatorial expression vector libraries (ExLib1 and ExLib2) were constructed using a randomization strategy that potentially could lead to variations in many of these sequence-related features and which would allow a pragmatic search of vector variants showing positive net effects on the level of soluble protein production. ExLib1 was constructed to encode all possible synonymous codons of an eight amino acid N-terminal extension of protein Z, fused to the N-terminal of an enhanced green fluorescent reporter protein (EGFP). In ExLib2, the same eight positions were randomized using an (NNG/T) degeneracy code, which could lead to various effects on both the nucleotide and protein level, through the introduction of nucleotide sequences functional as e.g. alternative ribosome binding or translation initiation sites or as translated codons for an Nterminal extension of the target protein by a peptide sequence. Flow cytometric analyses and sorting of library cell cultures resulted in isolation of clones displaying several-fold increases in whole cell fluorescence compared to a reference clone. SDS-PAGE and western blot analyses verified that this was a result of increases (up to 24-fold) in soluble intracellular ZEGFP product protein content. Both position specific codon bias effects and the appearance of new ribosomal binding sites in the library sequences were concluded to have influenced the protein production. To explore the possibility of applying the same combinatorial library strategy for improving soluble intracellular production of heterologous proteins proven difficult to express in E. coli, three proteins with either bacterial (a transcriptional regulator (DntR)) or human (progesterone receptor ligand binding domain (PRLBD) and 11-β Hydroxysteroid dehydrogenase type I (11-β)) origin, were cloned into the ExLib2 library. Flow cytometric sorting of libraries resulted in isolation of DntR library clones showing increased soluble protein production levels and PR-LBD library clones with up to ten-fold increases in whole cell fluorescence, although the product under these conditions co-separated with the insoluble cell material. / QC 20100623

<p>Abstract</p><p>The aim of the present study was to increase the understanding of how the cell surface properties affect the performance of unit operations used in primary protein purification. In particular, the purpose was to develop, set up and apply methods for studies of cell surface properties and cell interactions.</p><p>A method for microbial cell surface fingerprinting using surface plasmon resonance (SPR) is suggested. Four different <em>Escherichia coli </em>strains were used as model cells. Cell surface fingerprints were generated by registration of the interaction between the cells and four different surfaces, with different physical and chemical properties, when a cell suspension was flown over the surface. Significant differences in fingerprint pattern between some of the strains were observed. The physical properties of the cell surfaces were determined using microelectrophoresis, contact angle measurements and aqueous two-phase partitioning and were compared with the SPR fingerprints. The generated cell surface fingerprints and the physical property data were evaluated with multivariate data analysis that showed that the cells were separated into individual groups in a similar way using principal component analysis plots (PCA).</p><p>Studies of the behaviour of the model cells on stirred cell filtration and in an interaction test with different expanded bed adsorption (EBA) adsorbents were performed. It could be concluded that especially one of the strains behaved differently. Differences in the properties of the model cells were indicated by microelectrophoresis and aqueous two-phase partitioning which to some extent correlated with observed differences in behaviour during filtration and in an interaction test with EBA adsorbents.</p><p>The impact of high-pressure homogenisation of <em>E. coli </em>cell extract was examined, with a lab scale and a pilot scale technique. The DNA-fragmentation, visualised with agarose gel electrophoresis, and the resulting change in viscosity was analysed. A short homogenisation time resulted in increased viscosity of the process solution that correlated with increased concentration of released non-fragmented DNA. With longer homogenisation time the viscosity decreased with increasing degree of DNA-fragmentation.</p><p>The results show that strain dependant cell surface properties of<em> E. coli</em> may have an impact on several primary steps in downstream processing.</p>

The contribution of Cys17 and surface-exposed histidine residues in rhG-CSF interaction with Cu(II), Ni(II) and Hg(II) ions chelated by Light Resistant Yellow 2KT-polyethylene glycol derivative was evaluated in aqueous two-phase systems composed of polyethylene glycol (PEG) and dextran. It was determined that His43, His52, His156 and His170 residues are involved in protein interaction with chelated Cu(II) ions. Protein interaction with chelated Ni(II) is governed by His52 and His170 residues, though Cys17 is also involved. The contribution of Cys17 side chain is dominant in the interaction between rhG-CSF and chelated Hg(II) ions. The direct interaction between chelated Hg(II) ions and the –SH group of protein was determined for the first time. Based on the study of the interaction between rhG-CSF and chelated metal ions, rhG-CSF was successfully refolded from inclusion bodies in aqueous two-phase systems PEG-dextran containing chelated Ni(II) or Hg(II) ions for the first time. The refolding of rhG-CSF (C17S) in these systems was more effective compared to that of intact rhG-CSF. The dependence of refolding efficiency of rhG-CSF (C17S) in two-phase systems containing chelated metal ions on the number of histidine mutations was evaluated. It was determined that the refolding efficiency of protein in the systems containing chelated Ni(II) is inversely proportional to the number of histidine mutations. The affinity of purified rhG-CSF (C17S) and its histidine mutants for... [to full text]

Disponibiliza??o do conte?do parcial, conforme Termo de Autoriza??o no trabalho. / Submitted by Jos? Henrique Henrique (jose.neves@ufvjm.edu.br) on 2016-12-21T19:05:57Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) wbiratan_cesar_macedo_oliveira_parcial.pdf: 128173 bytes, checksum: 9c9067430cf3b7ef9b7952182484b258 (MD5) / Approved for entry into archive by Rodrigo Martins Cruz (rodrigo.cruz@ufvjm.edu.br) on 2017-01-31T14:19:01Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) wbiratan_cesar_macedo_oliveira_parcial.pdf: 128173 bytes, checksum: 9c9067430cf3b7ef9b7952182484b258 (MD5) / Made available in DSpace on 2017-01-31T14:19:01Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) wbiratan_cesar_macedo_oliveira_parcial.pdf: 128173 bytes, checksum: 9c9067430cf3b7ef9b7952182484b258 (MD5) Previous issue date: 2016 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior (CAPES) / Funda??o de Amparo ? Pesquisa do Estado de Minas Gerais (FAPEMIG) / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico (CNPq) / Neste trabalho foi estudado o comportamento de extra??o do lant?nio em sistemas aquosos bif?sicos (SAB) e foi desenvolvido um novo m?todo hidrometal?rgico ambientalmente seguro para a extra??o seletiva de La a partir de lixiviado de baterias NiMH. O comportamento de extra??o do La foi avaliado verificando a influ?ncia dos seguintes par?metros: presen?a e concentra??o de diversos agentes extratantes (Alaranjado de xilenol, 1-(2-piridilazo)-2-naftol, Ditizona, 8-hidroxiquinolina, e 1,10-fenantrolina); pH (3,00, 6,00 e 9,00); eletr?lito (Li2SO4, Na2SO4, MgSO4, Na2C4H4O6 e Na3C6H5O7 ) e pol?mero (L64 e PEO1500) formadores do sistema. A efici?ncia de recupera??o foi avaliada atrav?s da an?lise da porcentagem de extra??o (% E) e as melhores condi??es de extra??o do analito foram obtidas no SAB formado por PEO1500 + Li2SO4 + H2O, pH = 6,00; empregando o extratante 1,10-fenantrolina, atingindo um valor m?ximo de %E = 74,1 %. Na sequencia o m?todo foi aplicado a um lixiviado de bateria NiMH. Os terras raras foram precipitados mediante ajuste de pH (2,50) e posteriormente solubilizado em solu??o de H2SO4 0,1 M. Ap?s 3 etapas sucessivas de extra??o foram obtidos altos valores de fator de separa??o (S) entre o analito e os concomitantes met?licos (SLa,Co = 6.3 x 103, SLa,Ni= 2,55 x 104, SLa,Fe= 1,15 x 103 and SLa,Ce= 30,9). Al?m disso, foi realizado um ensaio de stripping no qual 88,5% de La, em uma ?nica etapa, foi redisponibilizado sob a forma i?nica na FI para uma poss?vel etapa posterior de eletrodeposi??o. / Disserta??o (Mestrado) ? Programa de P?s-Gradua??o em Qu?mica, Universidade Federal dos Vales do Jequitinhonha e Mucuri, 2016. / In this work was investigated the extraction behavior of lanthanum in the aqueous two-phase system (ATPS) and new environmentally safe hydrometallurgical method was developed for the selective extraction of La from NiMH battery leachate. The extraction behavior of La was evaluated verifying the influence of the following parameters on the metals extraction: presence and concentration of the several extractants 1-(2-piridil-azo)-2-naphtol, dithizone, 8-hydroxyquinoline, 1-10 phenanthroline and xylenol oragne; pH (3.00, 6.00 and 9.0); ATPS-forming electrolyte ((Li2SO4, Na2SO4, MgSO4, Na2C4H4O6 e Na3C6H5O7); ATPS-forming polymer (L64 or PEO1500). The recovery efficiency was evaluated through analyzing the extraction percentage (%E) and the best conditions for analyte extraction were achieved for the PEO1500 + Li2SO4 ATPS, pH = 6.00, using 1-10 phenanthroline as extractant agente (%E = 74,1 %). In the following section, the method was applied to a real NiMH battery leachate. The rare earths were precipitated by pH adjustment (2.50), which was solubilized in 0,1 M H2SO4 solution. After 3 steps of successive extractions it was possible to obtain high separation factor (S) values between the analyte and mettalic concomitant (SLa,Co = 6.3 x 103, SLa,Ni = 2,55 x 104, SLa,Fe = 1,15 x 103 and SLa,Ce = 30,9). Moreover, a stripping assay was carried out and after one single step, 88.5% of lanthanum was available to a possible electro winning step.

Data de aprova??o ausente. / Submitted by Jos? Henrique Henrique (jose.neves@ufvjm.edu.br) on 2017-03-29T18:49:58Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) wagner_barbosa_souza.pdf: 2617971 bytes, checksum: 3d7e6b01cafdb0a61d93af504071663c (MD5) / Approved for entry into archive by Rodrigo Martins Cruz (rodrigo.cruz@ufvjm.edu.br) on 2017-04-24T16:36:52Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) wagner_barbosa_souza.pdf: 2617971 bytes, checksum: 3d7e6b01cafdb0a61d93af504071663c (MD5) / Made available in DSpace on 2017-04-24T16:36:52Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) wagner_barbosa_souza.pdf: 2617971 bytes, checksum: 3d7e6b01cafdb0a61d93af504071663c (MD5) Previous issue date: 2016 / Com o avan?o exponencial da ind?stria de eletr?nicos, o consumo destes produtos tamb?m est? crescendo largamente. Com isso, os descartes destes equipamentos tamb?m crescem na mesma propor??o. Por ano, no mundo, estima-se que mais de 25 milh?es de toneladas de lixo eletr?nico sejam descartadas. Neste trabalho foi desenvolvido um novo m?todo hidrometal?rgico ambientalmente seguro para a extra??o seletiva de cobre, n?quel e prata provenientes de res?duos de placas de circuito impresso (PCI), empregando Sistemas Aquosos Bif?sicos (SAB) como t?cnica de extra??o. O comportamento de extra??o dos ?ons met?licos Cu (II), Ni(II), Fe(III) e Ag(I)em SAB formado por copol?mero tribloco L64 + MgSO4 + H2O foi avaliado para a otimiza??o do m?todo, verificando a influ?ncia dos seguintes par?metros experimentais sobre a extra??o dos analitos: valores de pH do SAB (3,0; 6,0; 9,0 e 11,0); natureza e concentra??o dos agentes extratores 1-(2-piridil-azo)-2-naftol (PAN) para os estudo de extra??o do Cu(II), 1-nitroso-2-naftol (1N2N) e dimetilglioxima (DMG) para a extra??o do ?on Ni(II), tiocianato (SCN) e ditizona (Dz) para a extra??o da Ag(I). A efici?ncia de recupera??o seletiva dos metais foi avaliada por meio da an?lise da porcentagem de extra??o (%E) e do fator de separa??o (S) entre os ?ons de interesse. As melhores condi??es para extra??o seletiva foram: PAN [3,5 mmol.Kg-1] em pH = 6,0 em 6 etapas consecutivas para separa??o do Cu(II), DMG [5,00 mmol.Kg-1] em pH = 9,0 para o Ni(II) e SCN[5,20 mmol.Kg-1] em pH = 9,0 para a Ag(I) . Em todas as situa??es foram obtidos valores de fator de separa??o(S) entre o analito e os concomitantes met?licos maiores que 103. As condi??es ?timas obtidas foram aplicadas ao lixiviado de PCI para a extra??o de cobre, n?quel e prata de forma sequencial, obtendo-se altos valores de S entre o analito e os concomitantes met?licos (SCu,Ni= 1,46 x 103, SCu,Fe= 1,55 x 104, SCu,Ag= 1,59 x 104, SNi,Fe= 3,27 x 104, SNi,Ag= 3,47 x 104 eSAg,Fe= 4,80 x 103).Ap?s a extra??o de cada metal foi realizado um estudo de stripping em uma ?nica etapa, onde 89,5%, 92,5% e 82,5% de Cu(II), Ni(II) e Ag(I), respectivamente foram disponibilizados para a etapa de eletrodeposi??o. Portanto o m?todo utilizando SAB se mostrou eficiente e dentro dos princ?pios da qu?mica verde, pois utiliza componentes at?xicos, biodegrad?veis e recicl?veis e de r?pida separa??o de fases sem a forma??o de emuls?es est?veis. Sendo um m?todo alternativo para a extra??o liquido ? liquido tradicional. / Disserta??o (Mestrado) ? Programa de P?s-Gradua??o em Qu?mica, Universidade Federal dos Vales do Jequitinhonha e Mucuri, [2016]. / With the exponential advancement of electronic industry, the consumption of these products is also growing wide. With this, the drops of this equipment also grow at the same rate. A year in the world, it is estimated that more than 25 million tons of electronic waste are disposed.This work developed a new environmentally safe hydrometallurgical method for the selective extraction of copper, nickel and silver from waste printed circuit board (PCB) using Aqueous Two-Phase Systems (SAB) as extraction technique. The extraction behavior of metal ions Cu (II), Ni (II), Fe (III) and Ag (I) SAB formed by tri-block copolymer L64 + MgSO4 + H2O was evaluated for the optimization of the method by checking the influence of the following experimental parameters on the extraction of analytes: the SAB pH (3.0, 6.0, 9.0 and 11.0); nature and concentration of agents extractors 1- (2-pyridyl-azo) -2-naphthol (PAN) Cu for the extraction method (II), the 1-nitroso-2-naphthol (1N2N) and dimethylglyoxime (DMG) for ion extraction Ni (II), thiocyanate (SCN) and dithizone (Dz) for the extraction of Ag (I).The selective metal recovery efficiency was evaluated by the percentage extraction analysis (% E) and separation factor (S) between the ions of interest. The best conditions for selective extractions were NAP [3,5 mmol.Kg-1] at pH 6.0 in 6 consecutive stages for separation of Cu (II) DMG [5.00 mmol.Kg-1] pH = 9.0 for Ni (II) and SCN [5,20 mmol.Kg-1] at pH = 9.0 for Ag (I). In all cases they were obtained separation factor values (S) between the analyte and the larger metal Concomitant 103. The obtained optimum conditions were applied to the PCI leached copper extraction, nickel and silver sequentially, yielding if high S values between the analyte and the metal concomitant (SCU, Ni = 1.46 x 103, SCU, Fe = 1.55 x 104, SCU, Ag = 1.59 x 104, SNI, Fe = 3.27 x 104, SNI, Ag = 3.47 x 104 and SAg, Fe = 4.80 x 103).After extraction of each metal it was made a study stripping in one step, where 89.5%, 92.5% and 82.5% Cu (II), Ni (II) and Ag (I), respectively, were available for the electroplating step. Therefore, the method using BSA was efficient and within the principles of green chemistry, because it uses non-toxic, biodegradable and recyclable and fast phase separation without the formation of stable emulsions components. As an alternative method for liquid - liquid extraction traditional.

Made available in DSpace on 2016-06-02T19:55:27Z (GMT). No. of bitstreams: 1 3130.pdf: 1560393 bytes, checksum: 956158589ca7612292e905d81a005f93 (MD5) Previous issue date: 2010-06-09 / Universidade Federal de Sao Carlos / Clavulanic acid is a &#946;-lactam substance with low antibiotic activity. Nonetheless, it is an important medicine which acts as a potent &#946;-lactamase inhibitor. These enzymes catalyze the hydrolysis of &#946;-lactam ring of antibiotics, leaving them without antibiotic action. It is industrially produced with submerged cultures of Streptomyces clavuligerus, a filamentous bacterium. Extraction and purification studies have shown a very clear and defined course, when it comes to the stages that precede precipitation and crystallization. The extraction processes with ultrafiltration membranes, with organic solvents and extraction in aqueous two-phase systems (ATPS) have been studied whereas purification has been studied for processes which involve ion exchange. However, there are few works related to clavulanic acid aqueous two-phase system extraction and they are not conclusive. The present study proposed utilizing the aqueous two-phase system (ATPS) to purify the clavulanic acid and re-extract it with ion exchange adsorption, which might provide information for further studies on coupled processes which operate continuously. The first technique evaluated to re-extract the clavulanic acid was the separation process with membranes, and its results showed a low separation between PEG and clavulanic acid. In a second step, the ion exchange chromatography technique with Amberlite IRA-400Cl and Streamline Q XL resins was used. It was evidenced by the ion exchange chromatography that the Amberlite IRA- 400Cl resin makes the process of re-extraction of clavulanic acid from the top phase possible and that the phosphate present in the top phase makes clavulanic acid adsorption difficult for both studied resins. Addition of ethanol, in order to precipitate the phosphate salts, made the re-extraction of clavulanic acid from the ATPS top phase by Streamline Q XL resin possible. The third step of the global process was the optimization of clavulanic acid extraction using the aqueous two-phase systems. The results showed that it is possible to obtain yields around 100% and a purification factor of 1.5 times for the clavulanic acid. Another characteristic analyzed was the clavulanic acid degradation velocity in the aqueous two-phase system; it was very high at the bottom phase which was rich in phosphate salts. Trials of continuous aqueous twophase system process were performed. This process was shown to be operationally viable. The set of results acquired in this study will allow the study and implementation xxv of a continuous process for the purification of clavulanic acid utilizing the aqueous twophase system. / O ácido clavulânico é uma substância &#946;-lactâmica com fraca atividade antibiótica, porém é um importante fármaco agindo como um potente inibidor de enzimas &#946;- lactamase. Estas enzimas que catalisam a hidrólise do anel &#946;-lactâmico dos antibióticos, deixando os sem ação antibiótica. O ácido clavulânico é produzido industrialmente por culturas submersas da bactéria filamentosa Streptomyces clavuligerus. Os trabalhos referentes à extração e purificação têm mostrado uma direção bastante clara e definida no sentido das etapas envolvidas que precedem à precipitação e cristalização. Na linha de extração têm sido estudados os processos com membranas de ultrafiltração, processos de extração com solventes orgânicos e extração em sistemas de duas fases aquosas (SDFA), enquanto que a purificação tem sido estudada por processos envolvendo a troca iônica. No entanto, são poucos os trabalhos relacionados com a extração do ácido clâvulanico por sistema de duas fases aquosas, não sendo estes ainda conclusivos. No presente trabalho foi proposto utilizar o SDFA para purificar o ácido clavulânico e re-extraí-lo através da adsorção por troca iônica, fornecendo subsídios para futuros estudos de processos conjugados operando de forma contínua. A primeira técnica estudada para re-extração do ácido clavulânico do SDFA foi o processo de separação com as membranas de polisulfona de microfiltração e ultrafiltração operando em escoamento tangencial, cujos resultados mostraram baixa separação entre PEG e ácido clavulânico. Numa segunda etapa utilizou-se técnica de cromatografia de troca iônica com as resinas Amberlite IRA-400Cl e Streamline Q XL. Para cromatografia de troca iônica ficou evidenciado que a resina Amberlite IRA-400Cl possibilita o processo de re-extração do ácido clavulânico da fase de topo e que o fosfato presente na fase de topo dificulta adsorção do ácido clavulânico para ambas as resinas estudadas. Adição do etanol para precipitação dos sais de fosfato possibilitou a re-extração do ácido clavulânico da fase de topo do SDFA pela resina Streamline Q XL. Como terceira etapa do processo global foi realizada a otimização da extração do ácido clavulânico através dos sistemas de duas fases aquosas, os resultados mostraram que é possível obter rendimento próximo a 100% e um fator de purificação de 1,5 vezes para o ácido clavulânico. Outro fator analisado foi a velocidade de degradação do ácido clavulânico no sistema de duas fases aquosas; esta se mostrou bastante alta na fase de fundo rica em sais de fosfato. Os ensaios do processo do sistema de duas fases aquosas contínuo foram realizados, sendo que esse processo mostrou-se operacionalmente viável. O conjunto de xxiii resultados obtidos neste trabalho permitirão o estudo e implementação de um processo contínuo para a purificação do ácido clavulânico utilizando o sistema de duas fases aquosas.