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.

Submitted by Fabio Sobreira Campos da Costa (fabio.sobreira@ufpe.br) on 2017-07-28T13:17:05Z No. of bitstreams: 2 license_rdf: 811 bytes, checksum: e39d27027a6cc9cb039ad269a5db8e34 (MD5) DISSERTAÇÃO.pdf: 1904426 bytes, checksum: 881aba48363b69adb462f39f38edbbe8 (MD5) / Made available in DSpace on 2017-07-28T13:17:05Z (GMT). No. of bitstreams: 2 license_rdf: 811 bytes, checksum: e39d27027a6cc9cb039ad269a5db8e34 (MD5) DISSERTAÇÃO.pdf: 1904426 bytes, checksum: 881aba48363b69adb462f39f38edbbe8 (MD5) Previous issue date: 2016-02-23 / As proteases são enzimas com a capacidade de hidrolisar proteínas em peptídeos menores ou aminoácidos livres, sendo essenciais para animais, plantas e micro-organismos devido à sua atuação na regulação metabólica. As proteases são utilizadas com diversas finalidades, no processo industrial da fabricação de detergentes, na indústria farmacêutica e de alimentos, além de ser utilizada na recuperação e aproveitamento de resíduos e subprodutos. Em virtude da grande importância das proteases este trabalho teve como objetivo comparar a produção de proteases produzidas por Mucor subtilissimus UCP 1262 em fermentação em estado sólido (FES) e submersa (FS), bem como extrair em Sistema de duas fases aquosas (SDFA) PEG/Fosfato, as colagenases oriundas de ambas as fermentações. O meio de produção da FES e FS no qual o micro-organismo foi cultivado era constituído principalmente de farelo de soja e farinha de soja. As determinações enzimáticas e dosagem proteica foram realizadas após 72h de fermentação. Para montagem do SDFA foram realizados dois planejamentos fatoriais 23, o primeiro planejamento foi realizado com amostras da FES e o segundo com amostras da FS. Neste sistema foi analisada a influência de três variáveis no processo de extração: massa molar do PEG (200, 550 e 1000 g/mol), concentração do PEG (17,5; 20 e 22,5%) e concentração do sal fosfato de sódio (15; 17,5 e 20%). A maior produção de proteases (362,66 U/ml) ocorreu na FES, enquanto que na FS obteve-se apenas 26,33 U/ml. Dentre as atividades proteásicas específicas: colagenolítica, fibrinolítica e queratinolítica, os melhores resultados foram obtidos para a atividade colagenolítica, sendo esta de: 179,81 U/ml, em FES. A colagenase presente no extrato bruto obtida nos processos fermentativos foram particionadas para fase rica em PEG do SDFA. O maior valor para a variável resposta Fator de purificação (FP=3,49) foi obtido no sistema que utilizou o extrato obtido por FES. Com base nas condições estudadas, os dois sistemas mostraram-se viáveis para a extração de colagenase, pois além de ser um processo que pode ser utilizado em larga escala é constituído por componentes de baixo custo e as condições utilizadas no SDFA favoreceram a extração desta enzima. Todavia, a extração da colagenase oriunda da FES foi mais promissora em virtude da maior concentração da enzima de interesse encontrada nesse tipo de fermentação. / Proteases are enzymes with the ability to hydrolyze proteins into smaller peptides and free amino acids. They are vital for animals, plants and micro-organisms due to their role in metabolic regulation. Proteases have been used in various purposes, in the industrial process of detergents, pharmaceutical and food industry, as well as being used in the recovery and utilization of waste and by-products. Due to their economic feasibility and great medical and farmaceutical importance this study aimed to compare the production of proteases produced by Mucor subtilissimus UCP 1262 in solid state fermentation (SSF) and submerged fermentation (SF) as well as extract collagenolytic proteases using Aqueous two-phase system (ATPS) -PEG/Phosphate from both fermentations. The medium composition for the fungal fermentation in SSF and SF was based in soybean flour. Enzymatic determinations and protein levels were performed after 72 hours of fermentation. To mount the ATPS were two 23 factorial design, the first planning was carried out with samples of SSF and the second with samples of SF. In this system was analyzed the influence of three variables in the extraction process: PEG molar mass (200, 550 and 1000), the PEG concentration (17,5; 22,5 and 20%) and sodium phosphate salt concentration (15; 17,5 and 20%). The higher proteolytic activity (362,66 U/ml) was produced using SSF, while in the FS was obtained 26,33 U/ml. Among the specific proteolytic activities: collagenolytic, fibrinolytic and keratinolytic, the best results were obtained for the collagenolytic activity, this being: 179,81 U / ml in the SSF. The Collagenase present in the crude extract obtained in the fermentative processes partitioned preferencially to the PEG-rich phase. The highest value for the variable response Purification Factor (PF = 3,49) was obtained in the system that used SSF crude extract. According with the showed results, both extraction systems seemed to be feasible for collagenase extraction, as well as being a process that can be used in large scale, constituted by low cost components and conditions used in this ATPS favored the enzyme extraction. Furthermore, the collagenase extraction from the SSF was more promising because of the higher interest enzyme concentration found in this type of fermentation.

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

Abstract 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. A method for microbial cell surface fingerprinting using surface plasmon resonance (SPR) is suggested. Four different Escherichia coli 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). 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. The impact of high-pressure homogenisation of E. coli 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. The results show that strain dependant cell surface properties of E. coli may have an impact on several primary steps in downstream processing. / QC 20101129

No description available.

A utilização de resíduos agrícolas visando à produção de insumos por via biotecnológica tem se mostrado importante uma vez que estes resíduos são fontes renováveis de carbono. A fração hemicelulósica destes resíduos apresenta como componente principal a xilose, que pode ser utilizada como substrato em processos de bioconversão para a obtenção de produtos com valor agregado. Um destes produtos é a enzima glicose-6-fosfato desidrogenase (G6PD), primeira enzima da via das pentoses fosfato que pode ser utilizada como reagente analítico em análises quantitativas, sobretudo em estudos bioquímicos e médicos. O presente trabalho visou estudar o processo de purificação dessa enzima empregando a extração em sistemas de duas fases aquosas convencional (sem integração) e integrado ao rompimento celular, em duas escalas, reduzida e ampliada. A enzima foi produzida por Candida guilliermondii FTI 20037 cultivada em meio constituído de hidrolisado hemicelulósico de palha de arroz, sob condições pré-determinadas. Inicialmente, foram realizados ensaios para avaliar o efeito das variáveis volume de suspensão celular, velocidade de agitação do moinho de esferas de vidro e tempo sobre o rompimento das células. Os valores destas variáveis foram, então, estabelecidos em: 100 mL, 400 rpm e 25 minutos, respectivamente. Posteriormente, a influência da massa molar de PEG e comprimento de linha de amarração sobre a extração da G6PD foram investigados no sistema convencional (homogeneizado obtido a partir do rompimento celular, em presença ou ausência de fragmentos) e integrado (rompimento na presença dos componentes da extração), empregando-se a metodologia do planejamento experimental. Nos ensaios realizados em escala reduzida, sob condições otimizadas, alcançou-se um fator de purificação na fase rica em sal (FPf), ou fase fundo, de 2,8 e em maior escala, ou seja, em moinho de rompimento, de 1,3. Com isso, realizou-se o estudo cinético e termodinâmico empregando a enzima presente no homogeneizado antes da purificação e após purificada no processo integrado em escala reduzida, nas seguintes condições: TLL 40% e PEG 1500 mol/L. Os valores determinados para os parâmetros cinéticos foram Km, 0,07 e 0,05 mM, Vm, 34,8 e 19,1 U/L e dos parâmetros termodinâmicos &#916;G, -13,71 e -13,64 KJ/mol; &#916;H, -2,49 e -2,50 KJ/mol; &#916;S, 37,02 e 36,77 J/mol.K; Ea, 24,18 e 15,02 KJ/mol, da enzima presente no homogeneizado celular antes e após purificação, respectivamente. / The employment of agricultural residues aiming the attainment of biotechnological products has been shown its importance since these residues are renewable and low cost sources of carbon. The hemicellulosic fraction of these residues presents xylose as main component, which can be utilized as substrate for different bioconversion processes for the acquisition of high value products. As an example, glucose-6-phosphate dehydrogenase, the first enzyme of pentose phosphate pathway which can be used as analytical reagent in several quantitative analysis, mainly in biochemical and medical studies. The present work contemplated the study of glucose-6-phosphate (G6PD) purification process by a conventional aqueous two phase systems extraction and integrated with cell disruption, in two scales, reduced and increased. The enzyme was obtained from cells of Candida guilliermondii FTI 20037 grown in hemicellulosic rice straw hydrolysate, using conditions established in previous work. Initially, assays in bead mill were performed to determine the effect of cell suspension volume, agitation speed and time on cell disruption. The determined conditions were: 100 mL, 400 rpm and 25 minutes, respectively. After this, the influence of molar mass of PEG and tie line lenght (TLL) on the G6PD recovery were investigated in the conventional system (with previous disrupted cells, with or without cell fragments) and integrated (disruption in the presence of extraction components), using the experimental design methodology. In the reduced scale assays, in optimized conditions, a purification factor in salt rich phase (FPf), or bottom phase, of 2,8 was reached while in the increased scale, this means in bead mill, a FPf of 1,3 was attained. In addition, kinetic and thermodynamic studies were performed, employing the enzyme present in the homogenate before and after purification in reduced scale, in the following conditions: TLL of 40% and PEG 1500 mol/L. The established values for the kinetics parameters were Km, 0,07 and 0,05 mM, Vm, 34,8 and 19,1 U/L and of thermodynamics &#916;G, -13,71 and -13,64 KJ/mol; &#916;H, -2,49 and -2,50 KJ/mol; &#916;S, 37,02 and 36,77 J/mol.K; Ea, 24,18 and 15,02 KJ/mol, of the enzyme present in the homogenate before and after purification respectively.

A partição e purificação de ascorbato oxidase de abóbora (Cucurbita maxima) por extração líquido-líquido em sistema de duas fases aquosas (SDFA), pelos processos descontínuos e contínuos, utilizando coluna de discos rotativos perfurados (PRDC), foram estudadas. Foram utilizados planejamentos estatísticos para selecionar as variáveis significativas no processo descontínuo de purificação, e as variáveis estudadas foram massa molar e concentração do polietileno glicol (PEG), concentração de citrato, pH, concentração de NaCl, fator de diluição e massa total do sistema. Os melhores resultados (coeficiente de partição 1,72, recuperação 90,8% e aumento de pureza 3,12) foram obtidos nas seguintes condições: massa molar do PEG 20000 (g/mol), pH 6,0, concentração de PEG 25% (m/m) e concentração de citrato 10% (m/m). No valor de pH 6,0 e temperatura 35°C a ascorbato oxidase apresentou seus maiores valores de atividade, e manteve a estabilidade na faixa de pH 5,0 a 9,0 durante 36 horas e a temperaturas de até 40°C durante 1 hora. Experimentos também foram realizados para estimar as principais propriedades cinéticas e termodinâmicas da atividade e estabilidade da ascorbato oxidase, e esse estudo revelou que a enzima foi estável nas condições testadas. A PRDC mostrou um bom desempenho para extração da ascorbato oxidase em modo contínuo utilizando SDFA. A melhor condição operacional selecionada neste estudo foi selecionada com o auxílio de planejamentos estatísticos, sendo selecionadas as seguintes condições: massa molar do PEG 20000 (g/mol), concentração de PEG 20% (m/m), concentração de citrato 10% (m/m), velocidade de rotação dos discos de 80 rpm e velocidade da fase dispersa de 2 mL/min. Os melhores resultados em valores médios foram: coeficiente de partição 3,36, recuperação 152%, aumento de pureza 2,31, coeficiente de transferência de massa 0,045, eficiência de separação 43,7% e hold up 0,33. Os dados experimentais demonstram o potencial da aplicação do sistema de duas fases aquosas PEG/citrato para purificar a ascorbato oxidase utilizando coluna de discos rotativos perfurados. / The partition and purification of ascorbate oxidase from pumpkin (Cucurbita maxima) by liquid-liquid extraction in aqueous two-phase system (ATPS) by discontinuous and continuous process, using perforated rotating disc contactor (PRDC) was studied. Experimental designs were used to choose the significant variables for discontinuous process, and polyethylene glycol (PEG) molar mass and concentration, citrate concentration, pH, NaCl concentration, dilution factor and total mass of the system, were the variables studied. The better results (partition coefficient 1.72, recovery 90.8% and purification factor 3.12) were obtained with following conditions: PEG molar mass of 20000 g/mol, pH 6.0, PEG concentration of 25% (w/w) and citrate concentration of 10% (w/w). In the pH 6.0 and temperature of 35?C the ascorbate oxidase showed their high activity values and the enzyme was stable in the pH range of 5.0 to 9.0 during 36 hours and temperatures up to 40?C for 1 hour. Experiments were also conducted to estimate the main kinetic and thermodynamic properties of ascorbate oxidase activity and stability, and this study revealed the interesting stability of this enzyme. The PRDC showed a good performance for extracting in continuous mode using aqueous two-phase systems. The best operating condition was selected in this study for the extraction of ascorbate oxidase in the PRDC, and it was obtained with PEG molar mass of 20000 g/mol, PEG concentration of 20% (w/w) and citrate concentration of 10% (w/w), the disc rotational speed of 80 rpm and dispersed phase flowrate of 2 mL/min. The results in mean values were: partition coefficient 3.36, recovery 152%, purification factor 2.31, mass transfer coefficient 0.045, separation efficiency 43.7% and Hold up 0.33. The experimental data showed the potential application of aqueous two-phase systems PEG/citrate to purification ascorbate oxidase using perforated rotating disc contactor.

A partição e purificação de ascorbato oxidase de abóbora (Cucurbita maxima) por extração líquido-líquido em sistema de duas fases aquosas (SDFA), pelos processos descontínuos e contínuos, utilizando coluna de discos rotativos perfurados (PRDC), foram estudadas. Foram utilizados planejamentos estatísticos para selecionar as variáveis significativas no processo descontínuo de purificação, e as variáveis estudadas foram massa molar e concentração do polietileno glicol (PEG), concentração de citrato, pH, concentração de NaCl, fator de diluição e massa total do sistema. Os melhores resultados (coeficiente de partição 1,72, recuperação 90,8% e aumento de pureza 3,12) foram obtidos nas seguintes condições: massa molar do PEG 20000 (g/mol), pH 6,0, concentração de PEG 25% (m/m) e concentração de citrato 10% (m/m). No valor de pH 6,0 e temperatura 35°C a ascorbato oxidase apresentou seus maiores valores de atividade, e manteve a estabilidade na faixa de pH 5,0 a 9,0 durante 36 horas e a temperaturas de até 40°C durante 1 hora. Experimentos também foram realizados para estimar as principais propriedades cinéticas e termodinâmicas da atividade e estabilidade da ascorbato oxidase, e esse estudo revelou que a enzima foi estável nas condições testadas. A PRDC mostrou um bom desempenho para extração da ascorbato oxidase em modo contínuo utilizando SDFA. A melhor condição operacional selecionada neste estudo foi selecionada com o auxílio de planejamentos estatísticos, sendo selecionadas as seguintes condições: massa molar do PEG 20000 (g/mol), concentração de PEG 20% (m/m), concentração de citrato 10% (m/m), velocidade de rotação dos discos de 80 rpm e velocidade da fase dispersa de 2 mL/min. Os melhores resultados em valores médios foram: coeficiente de partição 3,36, recuperação 152%, aumento de pureza 2,31, coeficiente de transferência de massa 0,045, eficiência de separação 43,7% e hold up 0,33. Os dados experimentais demonstram o potencial da aplicação do sistema de duas fases aquosas PEG/citrato para purificar a ascorbato oxidase utilizando coluna de discos rotativos perfurados. / The partition and purification of ascorbate oxidase from pumpkin (Cucurbita maxima) by liquid-liquid extraction in aqueous two-phase system (ATPS) by discontinuous and continuous process, using perforated rotating disc contactor (PRDC) was studied. Experimental designs were used to choose the significant variables for discontinuous process, and polyethylene glycol (PEG) molar mass and concentration, citrate concentration, pH, NaCl concentration, dilution factor and total mass of the system, were the variables studied. The better results (partition coefficient 1.72, recovery 90.8% and purification factor 3.12) were obtained with following conditions: PEG molar mass of 20000 g/mol, pH 6.0, PEG concentration of 25% (w/w) and citrate concentration of 10% (w/w). In the pH 6.0 and temperature of 35?C the ascorbate oxidase showed their high activity values and the enzyme was stable in the pH range of 5.0 to 9.0 during 36 hours and temperatures up to 40?C for 1 hour. Experiments were also conducted to estimate the main kinetic and thermodynamic properties of ascorbate oxidase activity and stability, and this study revealed the interesting stability of this enzyme. The PRDC showed a good performance for extracting in continuous mode using aqueous two-phase systems. The best operating condition was selected in this study for the extraction of ascorbate oxidase in the PRDC, and it was obtained with PEG molar mass of 20000 g/mol, PEG concentration of 20% (w/w) and citrate concentration of 10% (w/w), the disc rotational speed of 80 rpm and dispersed phase flowrate of 2 mL/min. The results in mean values were: partition coefficient 3.36, recovery 152%, purification factor 2.31, mass transfer coefficient 0.045, separation efficiency 43.7% and Hold up 0.33. The experimental data showed the potential application of aqueous two-phase systems PEG/citrate to purification ascorbate oxidase using perforated rotating disc contactor.

A utilização de resíduos agrícolas visando à produção de insumos por via biotecnológica tem se mostrado importante uma vez que estes resíduos são fontes renováveis de carbono. A fração hemicelulósica destes resíduos apresenta como componente principal a xilose, que pode ser utilizada como substrato em processos de bioconversão para a obtenção de produtos com valor agregado. Um destes produtos é a enzima glicose-6-fosfato desidrogenase (G6PD), primeira enzima da via das pentoses fosfato que pode ser utilizada como reagente analítico em análises quantitativas, sobretudo em estudos bioquímicos e médicos. O presente trabalho visou estudar o processo de purificação dessa enzima empregando a extração em sistemas de duas fases aquosas convencional (sem integração) e integrado ao rompimento celular, em duas escalas, reduzida e ampliada. A enzima foi produzida por Candida guilliermondii FTI 20037 cultivada em meio constituído de hidrolisado hemicelulósico de palha de arroz, sob condições pré-determinadas. Inicialmente, foram realizados ensaios para avaliar o efeito das variáveis volume de suspensão celular, velocidade de agitação do moinho de esferas de vidro e tempo sobre o rompimento das células. Os valores destas variáveis foram, então, estabelecidos em: 100 mL, 400 rpm e 25 minutos, respectivamente. Posteriormente, a influência da massa molar de PEG e comprimento de linha de amarração sobre a extração da G6PD foram investigados no sistema convencional (homogeneizado obtido a partir do rompimento celular, em presença ou ausência de fragmentos) e integrado (rompimento na presença dos componentes da extração), empregando-se a metodologia do planejamento experimental. Nos ensaios realizados em escala reduzida, sob condições otimizadas, alcançou-se um fator de purificação na fase rica em sal (FPf), ou fase fundo, de 2,8 e em maior escala, ou seja, em moinho de rompimento, de 1,3. Com isso, realizou-se o estudo cinético e termodinâmico empregando a enzima presente no homogeneizado antes da purificação e após purificada no processo integrado em escala reduzida, nas seguintes condições: TLL 40% e PEG 1500 mol/L. Os valores determinados para os parâmetros cinéticos foram Km, 0,07 e 0,05 mM, Vm, 34,8 e 19,1 U/L e dos parâmetros termodinâmicos &#916;G, -13,71 e -13,64 KJ/mol; &#916;H, -2,49 e -2,50 KJ/mol; &#916;S, 37,02 e 36,77 J/mol.K; Ea, 24,18 e 15,02 KJ/mol, da enzima presente no homogeneizado celular antes e após purificação, respectivamente. / The employment of agricultural residues aiming the attainment of biotechnological products has been shown its importance since these residues are renewable and low cost sources of carbon. The hemicellulosic fraction of these residues presents xylose as main component, which can be utilized as substrate for different bioconversion processes for the acquisition of high value products. As an example, glucose-6-phosphate dehydrogenase, the first enzyme of pentose phosphate pathway which can be used as analytical reagent in several quantitative analysis, mainly in biochemical and medical studies. The present work contemplated the study of glucose-6-phosphate (G6PD) purification process by a conventional aqueous two phase systems extraction and integrated with cell disruption, in two scales, reduced and increased. The enzyme was obtained from cells of Candida guilliermondii FTI 20037 grown in hemicellulosic rice straw hydrolysate, using conditions established in previous work. Initially, assays in bead mill were performed to determine the effect of cell suspension volume, agitation speed and time on cell disruption. The determined conditions were: 100 mL, 400 rpm and 25 minutes, respectively. After this, the influence of molar mass of PEG and tie line lenght (TLL) on the G6PD recovery were investigated in the conventional system (with previous disrupted cells, with or without cell fragments) and integrated (disruption in the presence of extraction components), using the experimental design methodology. In the reduced scale assays, in optimized conditions, a purification factor in salt rich phase (FPf), or bottom phase, of 2,8 was reached while in the increased scale, this means in bead mill, a FPf of 1,3 was attained. In addition, kinetic and thermodynamic studies were performed, employing the enzyme present in the homogenate before and after purification in reduced scale, in the following conditions: TLL of 40% and PEG 1500 mol/L. The established values for the kinetics parameters were Km, 0,07 and 0,05 mM, Vm, 34,8 and 19,1 U/L and of thermodynamics &#916;G, -13,71 and -13,64 KJ/mol; &#916;H, -2,49 and -2,50 KJ/mol; &#916;S, 37,02 and 36,77 J/mol.K; Ea, 24,18 and 15,02 KJ/mol, of the enzyme present in the homogenate before and after purification respectively.

Parmi les différentes méthodes de lutte contre les microorganismes pathogènes et/ou altérants en agroalimentaire, l’utilisation de bactéries lactiques (LAB) bioprotectrices s'avère être un outil prometteur pour la préservation des aliments. Ce travail de thèse collaboratif, entre l'équipe PAPC (AgroSup Dijon, Université de Bourgogne) et le laboratoire BioDyMIA (Université Lyon1-Isara Lyon), concerne l'étude de systèmes bioprotecteurs immobilisant des cellules entières de LAB dans une matrice polymérique d'alginate de sodium et de caséinate de sodium pour une activité ciblée contre Listeria spp. Dans un premier temps, la méthodologie mise en œuvre a consisté à sélectionner des souches de LAB bioprotectrices sur la base de leur activité antimicrobienne évaluée par la méthode de diffusion en milieu gélosé contre trois souches de Listeria spp. Quatre souches sur 19 ont ainsi été sélectionnées. Une caractérisation partielle des métabolites antimicrobiens produits par ces 4 souches a ensuite été réalisée en appliquant des traitements thermiques et enzymatiques aux surnageants de culture correspondants pour évaluer si ces traitements altéraient l’activité des métabolites antimicrobiens présents. Une purification et une identification partielle des actifs antimicrobiens de nature peptidique ont été réalisées uniquement pour la souche d'intérêt principale : Lactococcus lactis LAB3. Dans un second temps, une formulation de la matrice polymérique d’immobilisation des LAB sélectionnées a été choisie en réalisant le diagramme de phases du système aqueux alginate de sodium/caséinate de sodium : 1,5 % (m/m) d'alginate de sodium / 4 % (m/m) de caséinate de sodium / 20 % (m/m) bouillon MRS. Cette formulation a permis d'obtenir une matrice composée d’une phase continue riche en alginate et d’une phase dispersée riche en caséinate dans laquelle les cellules de LAB se localisent préférentiellement d’après les observations en microscopie de fluorescence confocale à balayage laser. Suite à l'inclusion des cellules de LAB dans ces matrices liquides et gélifiées d'alginate seul et d'alginate/caséinate, leur cultivabilité et leur activité anti-Listeria ont été suivies à 30°C pendant 12 jours. Ceci a révélé que la cultivabilité et l’activité antimicrobienne des cellules de LAB se maintiennent à des niveaux plus élevés dans les matrices d'alginate/caséinate que dans celles uniquement à base d’alginate. Ces matrices à base d’alginate et de caséinate apparaissent donc comme un système prometteur pour l'immobilisation de LAB bioprotectrices. Leur intérêt pour l’inclusion de LAB a pu être corrélé à leur viabilité et à la structure composite de cette matrice à base de protéines qui favoriserait la production et la libération des métabolites antimicrobiens / Among the various methods to control foodborne pathogenic and/or food spoilage microorganisms in food chain, bioprotective lactic acid bacteria (LAB) appear to be promising tools for food biopreservation. This collaborative study, between PAPC (Agrosup Dijon, University of Burgundy) and BioDyMIA (University Lyon1-Lyon Isara) laboratories, concerned the development of sodium alginate/sodium caseinate polymeric matrices intended to entrap LAB cells selected for their anti-Listeria spp. activity. First, 4 LAB strains from 19 LAB strains were selected for their anti-Listeria spp. activity: this screening was performed by the method of agar diffusion against three Listeria spp strains. Then, antimicrobial metabolites produced by the selected LAB strains were partially characterized by assessing the effect of various thermal and enzymatic treatments on the anti-Listeria spp. activity of their culture supernatants. A partial purification and identification of antimicrobial active peptides produced by the main strain of interest (Lactococcus lactis LAB3) was also performed. A composition of the polymer matrix has been selected by performing the phase diagram of sodium alginate/sodium caseinate system: 1.5% (w/w) sodium alginate / 4% (w/w) of caseinate sodium / 20% (w/w) MRS broth. This formulation provides a rich alginate continuous phase and a rich caseinate dispersed phase in which LAB cells localize according to the study by confocal microscopy. LAB cells were immobilized in liquid and gelled matrices of alginate and alginate/caseinate. Culturability and anti-Listeria activities were measured during a storage at 30°C for 12 days. The alginate/caseinate matrices were more effective in better maintaining LAB cells cultivability and their antimicrobial activity than alginate matrix. This effectiveness seemed correlated with cell viability and the dispersion-like structure of the protein-based system which enhance production and release of antimicrobial metabolites. Thus, this type of polymeric matrix appeared as a promising immobilization system of bioprotective LAB