Made available in DSpace on 2015-05-08T14:59:43Z (GMT). No. of bitstreams: 1 arquivototal.pdf: 3231556 bytes, checksum: dbeb38d3575cc8d87938d6de158f2b05 (MD5) Previous issue date: 2012-04-13 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / This work has as objective to realize out a theoretical and experimental study of the specific heat and the sorption kinetics in a fixed reactor. The specific heat experiment was conducted at materials laboratory at UFPB and experimental bench of the kinetic of sorption in fixed reactor was mounted in the solar energy laboratory, both located in the city João Pessoa. The experimental apparatus consisting of fixed bed filled with silica gel and subjected to a flow of moist air. For the theoretical study realized in a fixed bed, was developed a computer code with the purpose of identifying various physical and geometrical parameters, which could influence the dynamics of sorption, allowing simulating the processes of dehumidification and regeneration in fixed beds. The computational code was validated comparing the numerical results with experimental results. Studies were also performed to determine the specific heat in the adsorbed phase using the DSC method and from these results, it was determined an equation general through least squares regression for the calculation of the specific heat in the adsorbed phase. The Results were presented about pressure drop in the adsorptive bed, the mass profile of adsorbed to investigate the time of saturation of the bed, the temperature profiles and absolute humidity at the output of a fixed bed and also the experimental results of the specific heat the adsorbed phase with various concentrations . With this study we can conclude that the computer code developed is an important tool for the design of fixed bed adsorption, because without this tool would require a very high cost to modify the configuration in a fixed bed, defining a better choice of experimental setup. / O presente trabalho tem como objetivo realizar um estudo teórico e experimental do calor específico e da cinética de sorção em reator fixo. O experimento do calor específico foi realizado no laboratório de materiais da UFPB e a bancada experimental da cinética de sorção em reator fixo foi montada no Laboratório de Energia Solar (LES), ambos, localizados na cidade de João Pessoa. O aparato experimental consiste em um leito fixo preenchido com sílica gel e submetido a um fluxo de ar úmido. Para o estudo teórico realizado no leito fixo, foi desenvolvido um código computacional com o intuito de se verificar diversos parâmetros físicos e geométricos, que poderiam influenciar na dinâmica de sorção, permitindo simular processos de desumidificação e regeneração em leitos fixos. O código computacional desenvolvido foi validado, comparando-se os resultados obtidos numericamente com os resultados experimentais. Foram também realizados estudos para determinar o calor específico na fase adsorvida utilizando o método DSC e a partir desses resultados, determinou-se uma equação geral através de uma regressão por mínimos quadrados, para o cálculo do calor específico na fase adsorvida. Foram apresentados resultados da perda de carga no leito adsortivo, perfis de massa adsorvida para investigar o tempo de saturação do leito, perfis de temperatura e umidade absoluta na saída do leito fixo, como também, resultados experimentais do calor específico na fase adsorvida com várias concentrações. Com este estudo podê-se concluir que o código computacional desenvolvido é uma ferramenta importante para o dimensionamento de leitos fixos adsortivos, pois sem esta ferramenta seria necessário um custo muito alto para modificar a configuração no leito fixo, podendo-se com ele definir uma melhor opção de montagem experimental.

Orientadores: Telma Teixeira Franco, Reginaldo Guirardello / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia Quimica / Made available in DSpace on 2018-07-28T22:33:20Z (GMT). No. of bitstreams: 1 Santos_EveraldoSilvinodos_D.pdf: 4348712 bytes, checksum: f3506ac5746394a5083752c78e0b99b3 (MD5) Previous issue date: 2001 / Resumo: O presente trabalho refere-se ao uso da técnica de adsorção em lejto expandido para recuperar e purificar enzimas. Aspectos fundamentais da adsorção em leito expandido são abordados, utilizando lisozima e soro albumina bovina como proteínas modelo, e as enzimas, gliceraldeído-3-fosfato desidrogenase de Saccharomyces cerevisae, xilanase (extrato comercial) e quitosanase produzida por Bacil/us cereus. A avaliação da influência do uso de dois distribuidores, poroso e do tipo prato perfurado, mostrou que o distribuidor do tipo prato perfurado favoreceu mais à adsorção em leito expandido e que os adsorventes Streamline@ SP e Streamline@ DEAE possuem uma ampla distribuição de tamanho de partículas favorecendo ao fenômeno de segregação (Capítulo 2 - Artigo publicado no IEX 2000 (Cambridge/UK)). O uso de um processo integrado para recuperar e purificar a enzima intracelular Gliceraldeído 3-fosfato desidrogenase mostrou-se bem sucedido. O desempenho hidrodinâmico e cromatográfico foi estudado usando um adsorvente de estrutura pelicular (Pellicular) e dois adsorventes porosos comerciais (Stream1ine e Macrosorb) . Os resultados mostraram que o adsorvente Pellicular apresentou as melhores propriedades hidrodinâmicas e cromatográficas. (Capítulo 3 - Artigo em parceria e que será submetido à periódico internacional). O estudo da influência do uso de uma altura do leito empacotado, de 0,050 m e de 0,075 m na ALE operando-se em 10% da curva de ruptura, mostrou que uma altura de 0,075 m foi mais eficiente. Para o sistema lisozima - Streamline@ DEAE o rendimento aumentou com o aumento da velocidade linear enquanto que para o sistema BSA - Streamline@ SP esse fato não foi observado. Neste caso, para o sistema BSA - Streamline@ SP, a transferência de massa parece ser limitada pela menor densidade de carga acarretando assim em menores valores de eficiência em 10% de ruptura. (Capítulo 4 - Artigo aceito para publicação no periódico Biosepara_ion). O estudo da influência do conteúdo de células na adsorção em leito expandido mostrou que quando foi utilizado o extrato com o conteúdo de 5% de células (peso úmido), em leito expandido, o desempenho da purificação foi comprometido. (Capítulo 5 - Artigo aceito para publicação no periódico Joumal of Chromatography A). A adsorção de quitosanase de um caldo de fermentação de Baci/lus cereus em leito empacotado permitiu à obtenção de um pico com um fator de purificação de 7,6 e uma recuperação de 67,4% que eluiu com 0,51 M de NaCL Valores muito próximos a estes foram obtidos com o uso da adsorção em leito expandido com ambos os caldos, bruto e clarificado. Eletroforese em gel de poliacrilamida (SDS­P AGE) realizada para o tubo que exibiu a maior atividade, quando o leito foi no modo expandido e com células, mostrou a presença de duas bandas. Este fato sugere três possibilidades, a existência de duas quitosanases, a presença de um proteína contaminante ou a existência de uma quitosanase com duas sub-unidades (dímera). Entretanto, grande parte dos contaminantes foram separados da quitosanase em uma única etapa. (Capítulo 6 - Artigo que será submetido à um periódico internacional) / Abstract: This work deals with the application of Expanded Bed Adsorption (EBA) to recovery and purify enzymes. Model proteins, lisozyme and bovine serum albumin (BSA), as well as some enzymes, glyceraldehyde 3-phosphate dehydrogenase (G3PDH) from baker's yeast, xylanase (from a commercial extract) and chitosanase from Bacil/us cereus were used to study the EBA background and application. The influence of two distributor (porous and perfurated plate) showed that the perfurated plate distributor was more favorable for the EBA. The Strearnline@ SP and the Strearnline@ DEAE adsorbents have a wide size distribution favourable for the segregation phenomena. (Chapter 2 - artic1e published in IEX 2000 (Carnbridge/UK». An integrated process was successful to recover and to purify an intracellular enzyme, Glyceraldehyde 3-Phosphate Dehydrogenase (G3PDH). Performance of the hydrodynamic and chromatographic properties using a pellicular adsorbent (Pellicular) and two porous commercial adsorbents (Stream1ine and Macrosorb) showed that the former presented the best hydrodynarnic and chromatographic properties. (Chapter 3 - paper that wiil be submmited to an international journal). The influence of two settled bed height, 0.050 m and 0.075m, respectively, in the EBA operating at 10% of the breaktrough curve, showed that the former was more efficient. For the lisozyme - Strearnline@ DEAE system yield increased with the increase of the velocity while for the BSA - Strearnline@ SP system this behaviour was not observed. In this case, mass transfer seems to be limited problably due to its lower charge density. (Chapter 4 - paper accepted for publication in the Bioseparation Journal). The influence of cell contents in the EBA using a 5% (wet weight) cell content showed that the purification perfonnance was hampered. (Chapter 5 ­paper accepted for publication in the Journal ofChromatography A). The chitosanase adsorption of fermentation broth from Bacillus cereus, in packed mode, showed a chitosanase peak that eluted with 0.51 M NaCl, in this case a 7.6-fold purification factor with 67.4% of activity recovery were obtained. Similar values were obtained for both c1arified and unc1arified fermentation broth using the bed in the expanded mode. Experiment in expanded mode showed that two bands were present in the peak showing the highest activity, this suggests three possibilities, the existence of two chitosanases, the existence of a contaminant protein or the existence of a chitosanase with two sub-units. However, it was possible to separate the chitosanase from the-main contaminants in just one step. (Chapter 6 - paper that will be submitted to an international journal) / Doutorado / Desenvolvimento de Processos Químicos / Doutor em Engenharia Química

Global warming along with the climate change derived from the World's demand for energy are among the greatest challenges to our society. To tackle climate change issue, research must focus on proposing practical approaches for carbon emissions reduction and environmental remediation. This thesis focuses on carbon dioxide separation mainly from flue gases (major sources of carbon dioxide emissions) using metal organic frameworks (MOFs) to reduce its impact on the global warming hence the climate change. MOFs are a class of crystalline porous adsorbents with structures that attract CO2 selectively and store it in their porous frameworks. Over the course of this PhD research, the fundamental aspects of these materials, as well as their practical applications, have been investigated. For example, the synthesis recipe of copper (II) benzene-1,3,5-tricarboxylate (CuBTC) MOF was improved to deliver a product of high yield ( > 89%) and free of by-product. Also, a mechanism study on the hydrothermal stability CuBTC MOF was carried out under simulated flue gas conditions and delivered the first experimental proof of the decomposition mechanism of CuBTC MOF caused by the water vapour. The fundamental understanding of the stability of materials then motivated the research into the development of a facile method of using an economic functional dopant (i.e. glycine) to strengthen the structure of CuBTC MOF (completely stable towards water vapour), as well as to improve the selectivity of resulting materials to CO2 (by 15% in comparison to the original CuBTC MOF). The suitability of the CuBTC MOF for fixed bed adsorption processes was also assessed using a combined experimental and process simulation method. In addition to the experimental approaches, molecular simulation based on grand canonical Monte Carlo method was also used to understand the effect of structural defects of MOFs on the CO2 adsorption isotherms.

<p>The aim of this work was to develop a production process for the enzyme xyloglucan <i>endo</i>-transglycosylase from <i>Populus tremula x tremuloides</i> (<i>Ptt</i>XET16-34). The natural transglycosylating activity of this enzyme has previously been employed in a XET-Technology. This chemo enzymatic method is useful for biomimetic modification of cellulose surfaces and holds great potential for industrial applications. Thus, it requires that the XET-enzyme can be produced in larger scale.</p><p>This work also shows how the wildtype <i>Ptt</i>XET16-34 was modified into a glycosynthase. By mutation of the catalytic nucleophile into an alanine, glycine or serine residue, enzymes capable of synthesising defined xyloglucan fragments were obtained. These defined compounds are very valuable for further detailed studies of xyloglucan active-enzymes, but are also useful in molecular studies of the structurally important xyloglucan-cellulose interaction.</p><p>A heterologous production system for <i>Ptt</i>XET16-34 was previously developed in the methylotrophic yeast Pichia pastoris. A methanol-limited fed-batch process was also previously established, but the yield of active XET was low due to proteolysis problems and low productivity. Therefore, two alternative fed-batch techniques were investigated for the production of <i>Ptt</i>XET16-34: a temperature-limited fed-batch (TLFB) and an oxygen-limited high-pressure fed-batch (OLHPFB).</p><p>For the initial recovery of XET after the fermentation process, two different downstream processes were investigated: expanded bed adsorption (EBA) and cross-flow filtration (CFF).</p>

With the increased demands of therapeutic proteins, there is going to be a need for new purification technologies which have high throughput, high yield and high resolution. Three purification technologies were explored as potential new technology to isolate recombinant and endogenous milk proteins: Expanded bed adsorption chromatography(EBAC) combined with hydrophobic interaction chromatography(HIC), Recycle continuous flow electrophoresis(RCFE) and Free flow isoelectric focusing(FFIEF). The first process(EBAC/HIC) used with Zn2+ as a selective precipitating agent, purified recombinant human protein C(rhPC) and IgG(contaminated with less than 1% IgA) from swine milk with high resolution and high yield while processing about 10-20 grams in a single operation. The second process(RCFE) was able to isolate the active sub-populations of rhPC from major milk contaminants( - and -pig casein) as wells as from the inactive sub-populations of rhPC. RCFE was able to process 1.5g total protein per hour on a small scale and is currently being researched to process 1kg total protein per hour. The third and final purification process(FFIEF) sub-fractionated 100mg of immuno-purified rhPC into 50 fractions. The FFIEF was able to produce a linear pH gradient over the range of 3-10 using 2% ampholytes. The fractionated rhPC showed differing degrees of activity that resulted from the -carboxylated glutamic acids and the sialic acids. / Ph. D.

The aim of this work was to develop a production process for the enzyme xyloglucan endo-transglycosylase from Populus tremula x tremuloides (PttXET16-34). The natural transglycosylating activity of this enzyme has previously been employed in a XET-Technology. This chemo enzymatic method is useful for biomimetic modification of cellulose surfaces and holds great potential for industrial applications. Thus, it requires that the XET-enzyme can be produced in larger scale. This work also shows how the wildtype PttXET16-34 was modified into a glycosynthase. By mutation of the catalytic nucleophile into an alanine, glycine or serine residue, enzymes capable of synthesising defined xyloglucan fragments were obtained. These defined compounds are very valuable for further detailed studies of xyloglucan active-enzymes, but are also useful in molecular studies of the structurally important xyloglucan-cellulose interaction. A heterologous production system for PttXET16-34 was previously developed in the methylotrophic yeast Pichia pastoris. A methanol-limited fed-batch process was also previously established, but the yield of active XET was low due to proteolysis problems and low productivity. Therefore, two alternative fed-batch techniques were investigated for the production of PttXET16-34: a temperature-limited fed-batch (TLFB) and an oxygen-limited high-pressure fed-batch (OLHPFB). For the initial recovery of XET after the fermentation process, two different downstream processes were investigated: expanded bed adsorption (EBA) and cross-flow filtration (CFF). / <p>QC 20101108</p>

<p>The two techniques aqueoustwo-phase partitioning and expanded bed adsorption that bothare suitable for primary protein recovery were studied. Most ofthe work was focused on partition in aqueous two-phase systemsand in particular on the possibility to effect the partitionbehaviour by fusion of short peptide tags or protein domains tothe target protein.</p><p>The partitioning of fusionproteins between different variants of the domain tag Z and thenaturally occurring protein DNA Klenow polymerase were studiedin Breox/Reppal aqueous two-phase systems. Most studies wereperformed with cell homogenate. The Breox/Reppal system was infocus because if the fusion protein can be partitioned to theBreox-rich top phase the next step can be a thermoseparatingaqueous two-phase system. When the Breox phase is heated to50°C it switches from a one-phase system to a two-phasesystem resulting in an almost pure water rich top phase andhighly concentrated Breox-rich bottom phase. The Breox can thenbe reused and the protein recovered from the water phase. TheZ-domain was genetically modified in different ways to Z_basic1, Z_acid2and Z_trp12and fused to the Klenow protein to try toenhance partitioning to the Breox-rich phase. From theexperiments it was not possible to observe any effects on thepartition behaviour irrespectively of tested properties of thedomain tag. Despite the absence of domain tag effects highK-values, i.e. partition to the Breox-rich top phase, wereobserved in the Breox/Reppal system. However, the proteinK-values seemed to be rather sensitive to the cell homogenateload and showed a tendency to decrease with increased cellhomogenate load. Also increased phosphate concentration reducedthe K-values. The partitioning of cell debris also seemed todependent on the cell homogenate load. At higher homogenateload (<=20g DW/L) clear Breox-rich top phases were observedwith the cell debris collected in Reppal-rich bottomphases.</p><p>Two different tetrapeptides,AlaTrpTrpPro and AlaIleIlePro were inserted near the C-terminusof the protein ZZT0. The Trp-rich peptide unit stronglyincreased both the partitioning of ZZT0 into the poly(ethyleneglycol) (PEG)-rich phase in a PEG/potassium phosphate aqueoustwo-phase system and its retention on PEG and propylhydrophobic interaction chromatographic columns with potassiumphosphate as eluent in isocratic systems. Both the partitioningand the retention increased with increasing number of Trp-richpeptide units inserted into ZZT0. Insertion of Ile-richtetrapeptide units affected the partitioning and retention to amuch lesser extent. Partition and modelling data also indicateda folding of inserted Trp and Ile tetrapeptide units, probablyto minimise their water contact. It was also investigated howto predict the partitioning of proteins in isoelectricPEG/phosphate aqueous two-phase systems.</p><p>The capture ofß-galactosidase from<i>E. coli</i>cell homogentate (50g DW/L) by metal chelatexpanded bed adsorption was studied. These experiments showedthat capture, with a certain degree of selectivity, andclarification of ß-galactosidase could be achieved from acell homogenate. However, a rather low recovery of about 35 %was obtained at a capacity of 0.25mg/mL of gel. Thus, severalparameters remain to be optimised like the load buffercomposition and the cell homogenate load.</p><p><b>Keywords:</b><i>E. coli</i>, aqueous two-phase systems, fusion proteins,hydrophobic interaction chromatography, expanded bedadsorption, ß-galactosidase, Klenow polymerase, Z-domain,peptide tags</p>

Made available in DSpace on 2014-12-17T15:01:49Z (GMT). No. of bitstreams: 1 JorgeSC_TESE.pdf: 1730447 bytes, checksum: 39fc94239eeb6e994140c5c0f9a2cc23 (MD5) Previous issue date: 2010-06-22 / Expanded Bed Adsorption plays an important role in the downstream processing mainly for reducing costs as well as steps besides could handling cells homogenates or fermentation broth. In this work Expanded Bed Adsorption was used to recover and purify whey proteins from coalho cheese manufacture using Streamline DEAE and Streamline SP both ionic resins as well as a hydrophobic resin Streamline Phenyl. A column of 2.6 cm inner diameter with 30 cm in height was coupled to a peristaltic pump. Hydrodynamics study was carried out with the three resins using Tris-HCl buffer in concentration of 30, 50 and 70 mM, with pH ranging from 7.0 to 8.0. In this case, assays of the expansion degree as well as Residence Time Distribution (RTD) were carried out. For the recovery and purification steps, a whey sample of 200 mL, was submitted to a column with 25mL of resin previously equilibrated with Tris/HCl (50 mM, pH 7.0) using a expanded bed. After washing, elution was carried out according the technique used. For ionic adsorption elution was carried out using 100 mL of Tris/HCl (50 mM, pH 7.0 in 1M NaCl). For Hydrophobyc interaction elution was carried out using Tris/HCl (50 mM, pH 7.0). Adsorption runs were carried out using the three resins as well as theirs combination. Results showed that for hydrodynamics studies a linear fit was observed for the three resins with a correlation coefficient (R2) about 0.9. In this case, Streamline Phenyl showed highest expansion degree reaching an expansion degree (H0/H) of 2.2. Bed porosity was of 0.7 when both resins Streamline DEAE and Streamline SP were used with StremLine Phenyl showing the highest bed porosity about 0.75. The number of theorical plates were 109, 41.5 and 17.8 and the axial dipersion coefficient (Daxial) were 0.5, 1.4 and 3.7 x 10-6 m2/s, for Streamline DEAE, Streamline SP and Streamline Phenyl, respectively. Whey proteins were adsorved fastly for the three resins with equilibrium reached in 10 minutes. Breakthrough curves showed that most of proteins stays in flowthrough as well as washing steps with 84, 77 and 96%, for Streamline DEAE, Streamline SP and Streamline Phenyl, respectively. It was observed protein peaks during elution for the three resins used. According to these peaks were identified 6 protein bands that could probably be albumin (69 KDa), lactoferrin (76 KDa), lactoperoxidase (89 KDa), &#946;-lactoglobulin (18,3 KDa) e &#945;-lactoalbumin (14 KDa), as well as the dimer of beta-lactoglobulin. The combined system compound for the elution of Streamline DEAE applied to the Streamline SP showed the best purification of whey proteins, mainly of the &#945;-lactoalbumina / A adsor??o em leito expandido vem se destacando como uma t?cnica promissora dentro do downstream processing por ser de f?cil manuseio, baixo custo, diminuir etapas de processamento e utilizar o material particulado no seu estado natural. Portanto, o presente trabalho teve como objetivo recuperar e purificar prote?nas presentes no soro de queijo tipo coalho, atrav?s da t?cnica de adsor??o em leito expandido, utilizando resinas de troca ani?nica Streamline DEAE e troca cati?nica Streamline SP e intera??o hidrof?bica Streamline Phenyl,. Foi utilizada uma coluna de 2,6 cm de di?metro interno por 30 cm de altura, acoplada a uma bomba perist?ltica. Para o estudo do sistema foram realizados testes de hidrodin?mica e corridas de adsor??o, com as tr?s resinas, na presen?a de tamp?es Tris-HCl nas concentra??es 30, 50 e 70 mM, com pHs ajustados usando HCl para 7,0; 7,5 e 8,0. Para os testes hidrodin?micos foram estudados a expans?o do leito e a Distribui??o do Tempo de Resid?ncia (DTR). Na etapa de recupera??o e purifica??o, uma amostra de solu??o de soro de 200 mL foi aplicada, a temperatura ambiente, a uma coluna contendo resina (25 mL) previamente equilibrada em tamp?o Tris/HCl (50 mM e pH 7,0), ap?s lavagem efetuou-se a elui??o de acordo com o tipo de t?cnica utilizada. Dessa forma, para adsor??o com troca i?nica a elui??o ocorria com adi??o do eluente 100 mL Tris/HCl (50 mM, pH 7,0 em NaCl 1M). No caso de intera??o hidrof?bica, o eluente consistia de Tris/HCl (50 mM e pH 7,0). Os ensaios de adsor??o foram realizados com as resinas Streamline DEAE, Streamline SP e Streamline Phenyl e suas combina??es. Os resultados mostraram que para as condi??es em que foram realizados os ensaios fluidodin?micos e para o tipo de coluna utilizada, houve uma tend?ncia a linearidade, o coeficiente de correla??o (R2) foi da ordem de 0,9 e que a resina Streamline Phenyl obteve um maior grau de expans?o que as outras resinas, chegando a uma rela??o H0/H de 2,2. A porosidade do leito usando as resinas DEAE e SP foi de 0,70 e da resina Phenyl foi um pouco maior, em torno de 0,75. O n?mero de pratos te?ricos foi 109, 41,5 e 17,8 e o coeficiente de dispers?o axial (Daxial) foi de 0,5, 1,4 e 3,7 x 10-6 m2/s, para as resinas Streamline DEAE, Streamline SP e Streamline Phenyl, respectivamente. As prote?nas do soro s?o adsorvidas nas tr?s resinas e a concentra??o de prote?na em solu??o diminui rapidamente nos primeiros instantes do processo de adsor??o, sendo o equil?brio alcan?ado nos primeiros 10 minutos. Ao se aplicar o soro bruto sem tratamento para as tr?s resinas at? a satura??o (ruptura), embora exista adsor??o das prote?nas para essas resinas, perde-se grande parte dessas prote?nas nas etapas de passante e lavagem. Essas perdas somam 84, 77 e 96%, para as resinas Streamline DEAE, Streamline SP e Strealine Phenyl, respectivamente. Entretanto, pode-se recuperar 16, 23 e 4%, respectivamente, para as tr?s resinas. As tr?s resinas estudadas apresentaram picos de prote?nas na elui??o. De acordo com esses picos, foram identificadas 6 bandas de prote?nas. Provavelmente essas prote?nas sejam: albumina (69 KDa), lactoferrina (76 KDa) e lactoperoxidase (89 KDa), &#946;-lactoglobulina (18,3 KDa) e &#945;-lactoalbumina (14 KDa), d?mero da &#946; -lactoglobulina. Portanto, as resinas estudadas s?o compat?veis para serem utilizadas em leito expandido. O sistema formado pela elui??o da Streamline DEAE quando foi aplicada na resina Streamline SP, tende a uma melhor purifica??o das prote?nas do soro, principalmente da &#945;-lactoalbumina

The two techniques aqueoustwo-phase partitioning and expanded bed adsorption that bothare suitable for primary protein recovery were studied. Most ofthe work was focused on partition in aqueous two-phase systemsand in particular on the possibility to effect the partitionbehaviour by fusion of short peptide tags or protein domains tothe target protein. The partitioning of fusionproteins between different variants of the domain tag Z and thenaturally occurring protein DNA Klenow polymerase were studiedin Breox/Reppal aqueous two-phase systems. Most studies wereperformed with cell homogenate. The Breox/Reppal system was infocus because if the fusion protein can be partitioned to theBreox-rich top phase the next step can be a thermoseparatingaqueous two-phase system. When the Breox phase is heated to50°C it switches from a one-phase system to a two-phasesystem resulting in an almost pure water rich top phase andhighly concentrated Breox-rich bottom phase. The Breox can thenbe reused and the protein recovered from the water phase. TheZ-domain was genetically modified in different ways to Zbasic1, Zacid2and Ztrp12and fused to the Klenow protein to try toenhance partitioning to the Breox-rich phase. From theexperiments it was not possible to observe any effects on thepartition behaviour irrespectively of tested properties of thedomain tag. Despite the absence of domain tag effects highK-values, i.e. partition to the Breox-rich top phase, wereobserved in the Breox/Reppal system. However, the proteinK-values seemed to be rather sensitive to the cell homogenateload and showed a tendency to decrease with increased cellhomogenate load. Also increased phosphate concentration reducedthe K-values. The partitioning of cell debris also seemed todependent on the cell homogenate load. At higher homogenateload (&lt;=20g DW/L) clear Breox-rich top phases were observedwith the cell debris collected in Reppal-rich bottomphases. Two different tetrapeptides,AlaTrpTrpPro and AlaIleIlePro were inserted near the C-terminusof the protein ZZT0. The Trp-rich peptide unit stronglyincreased both the partitioning of ZZT0 into the poly(ethyleneglycol) (PEG)-rich phase in a PEG/potassium phosphate aqueoustwo-phase system and its retention on PEG and propylhydrophobic interaction chromatographic columns with potassiumphosphate as eluent in isocratic systems. Both the partitioningand the retention increased with increasing number of Trp-richpeptide units inserted into ZZT0. Insertion of Ile-richtetrapeptide units affected the partitioning and retention to amuch lesser extent. Partition and modelling data also indicateda folding of inserted Trp and Ile tetrapeptide units, probablyto minimise their water contact. It was also investigated howto predict the partitioning of proteins in isoelectricPEG/phosphate aqueous two-phase systems. The capture ofß-galactosidase fromE. colicell homogentate (50g DW/L) by metal chelatexpanded bed adsorption was studied. These experiments showedthat capture, with a certain degree of selectivity, andclarification of ß-galactosidase could be achieved from acell homogenate. However, a rather low recovery of about 35 %was obtained at a capacity of 0.25mg/mL of gel. Thus, severalparameters remain to be optimised like the load buffercomposition and the cell homogenate load. <b>Keywords:</b>E. coli, aqueous two-phase systems, fusion proteins,hydrophobic interaction chromatography, expanded bedadsorption, ß-galactosidase, Klenow polymerase, Z-domain,peptide tags / NR 20140805

No description available.