Membrane chromatography was investigated as an alternative method to packed-bed chromatography for protein recovery. The purification of lysozyme from egg white with Natrix adseptTM weak cation-exchange membranes was investigated under two different binding configurations: (1) a non-flow, static set-up with variable pH and sodium chloride (NaCl) concentrations during the binding and elution steps, and (2) a dynamic, cross-flow set-up with recycle at pH 7.5 and no NaCl addition during binding. The weak cation-exchange membrane consisted of a carboxylic acid-based, environmentally-responsive hydrogel layer bonded to a polymer matrix. Lysozyme was chosen to illustrate protein-membrane binding interactions due to its well-characterized nature and positive surface charge over a large pH range. For the static binding set-up, two sources of lysozyme were studied: pure lysozyme and egg whites treated with 60 % (v/v) ethanol (ESEW). Elution of bound protein was performed with 1 M NaCl under two pH strategies: binding and elution at a constant pH, and binding at pH 4.5 and variable elution pH. The highest maximum total protein binding capacity for pure lysozyme and ESEW was observed at pH 4.5 with no NaCl addition; however, poor total protein and lysozyme activity recovery were achieved during separation. As well, other egg white proteins, such as ovomucoid, were observed to bind to the membrane surface at pH 4.5, despite possessing similar charge polarity to the anionic membrane surface, indicating a non-electrostatic binding mechanism during operation below the membrane’s pKa (4.7). Based on the conditions tested, the highest total protein and lysozyme activity recovery was demonstrated for the separation of lysozyme from ESEW at pH 7.5 binding and elution and no NaCl addition. In the dynamic binding study, very high pure lysozyme dynamic binding capacity was achieved at 10 % breakthrough (167.3 mg/ml membrane for a 0.35 mg/ml lysozyme solution). The lysozyme dynamic binding capacity was 2.2 times greater than the static binding capacity under similar conditions, significantly higher than published results for other cation-exchange membranes. The separation of lysozyme from four lysozyme sources was tested: pure lysozyme, ESEW, and aqueous egg whites with (ASEW) and without (AEW) 100 mM NaCl. The highest lysozyme activity recovery during separation and lysozyme purity was achieved from the ESEW feed. Lysozyme separation from aqueous egg whites was not as effective, likely due to a high concentration of negatively-charged protein impurities fouling the surface of the membrane. Competitive binding to the membrane limited lysozyme binding and reduced the purity of the recovery elution stream. The application of feed-side pressure during the separation of ESEW produced a high purity, high recovery lysozyme elution stream with a significant reduction in processing time; however, protein aggregates were observed to form on the membrane surface, limiting the applicability of high-pressure operation and reducing protein functionality in the elution stream. The weak cation-exchange membrane system was shown to successfully separate out a target protein from a low concentration protein mixture through electrostatic interactions, and may be further applied to other protein systems.

Membrane chromatography was investigated as an alternative method to packed-bed chromatography for protein recovery. The purification of lysozyme from egg white with Natrix adseptTM weak cation-exchange membranes was investigated under two different binding configurations: (1) a non-flow, static set-up with variable pH and sodium chloride (NaCl) concentrations during the binding and elution steps, and (2) a dynamic, cross-flow set-up with recycle at pH 7.5 and no NaCl addition during binding. The weak cation-exchange membrane consisted of a carboxylic acid-based, environmentally-responsive hydrogel layer bonded to a polymer matrix. Lysozyme was chosen to illustrate protein-membrane binding interactions due to its well-characterized nature and positive surface charge over a large pH range. For the static binding set-up, two sources of lysozyme were studied: pure lysozyme and egg whites treated with 60 % (v/v) ethanol (ESEW). Elution of bound protein was performed with 1 M NaCl under two pH strategies: binding and elution at a constant pH, and binding at pH 4.5 and variable elution pH. The highest maximum total protein binding capacity for pure lysozyme and ESEW was observed at pH 4.5 with no NaCl addition; however, poor total protein and lysozyme activity recovery were achieved during separation. As well, other egg white proteins, such as ovomucoid, were observed to bind to the membrane surface at pH 4.5, despite possessing similar charge polarity to the anionic membrane surface, indicating a non-electrostatic binding mechanism during operation below the membrane’s pKa (4.7). Based on the conditions tested, the highest total protein and lysozyme activity recovery was demonstrated for the separation of lysozyme from ESEW at pH 7.5 binding and elution and no NaCl addition. In the dynamic binding study, very high pure lysozyme dynamic binding capacity was achieved at 10 % breakthrough (167.3 mg/ml membrane for a 0.35 mg/ml lysozyme solution). The lysozyme dynamic binding capacity was 2.2 times greater than the static binding capacity under similar conditions, significantly higher than published results for other cation-exchange membranes. The separation of lysozyme from four lysozyme sources was tested: pure lysozyme, ESEW, and aqueous egg whites with (ASEW) and without (AEW) 100 mM NaCl. The highest lysozyme activity recovery during separation and lysozyme purity was achieved from the ESEW feed. Lysozyme separation from aqueous egg whites was not as effective, likely due to a high concentration of negatively-charged protein impurities fouling the surface of the membrane. Competitive binding to the membrane limited lysozyme binding and reduced the purity of the recovery elution stream. The application of feed-side pressure during the separation of ESEW produced a high purity, high recovery lysozyme elution stream with a significant reduction in processing time; however, protein aggregates were observed to form on the membrane surface, limiting the applicability of high-pressure operation and reducing protein functionality in the elution stream. The weak cation-exchange membrane system was shown to successfully separate out a target protein from a low concentration protein mixture through electrostatic interactions, and may be further applied to other protein systems.

Proteomic studies at the single molecular level could provide better understanding of the protein’s behaviour and the effects of its interactions with other biomolecules. This could have an impact on drug development methods, disease diagnosis, and targeted therapy. Aperture assisted optical trapping is a proven technique for isolating single proteins in solution without the use of tethers or labels, and without denaturing them. Thus enabling studies of protein-protein interactions, protein-small molecule interactions, and protein-DNA interactions. In this work, double nanohole (DNH) optical tweezers were used to analyze the protein composition of heterogeneous mixtures. The trapped proteins were grouped by molecular mass based on two metrics: standard deviation of the trapping laser intensity fluctuations, and the time constant of the autocorrelation function of these fluctuations. The quantitative analysis is demonstrated first for two separate standard-size proteins, then for a mixed solution of both. Finally, the approach is applied to real unprocessed egg white solution. The results correspond well with the known protein composition of egg white found in the literature. The DNH optical tweezers’ ability to distinguish proteins in unpurified heterogeneous mixtures, can progress this technique to the next level, allowing for single biomolecular studies of unprocessed physiological solutions like blood, urine, or saliva. / Graduate

No description available.

Orientadores: Rosiane Lopes da Cunha, Florencia Cecilia Menegalli / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-05T17:14:21Z (GMT). No. of bitstreams: 1 Christ_Divair_D.pdf: 2473666 bytes, checksum: ca400b2519c40ad806addec20990ae8c (MD5) Previous issue date: 2006 / Resumo: O objetivo deste estudo foi avaliar o processo de secagem da clara de ovo utilizando um secador de leito de jorro fluidizado bidimensional com esferas de vidro como inertes. Um planejamento fatorial completo 24 foi realizado, com 3 pontos centrais e oito axiais, utilizando-se as seguintes variáveis independentes: temperatura (55-95 °C) e vazão total do ar de secagem (97,1-138,3 m³/h), razão entre vazão de ar de jorro e ânulo (0,6-1,4) e vazão de alimentação de pasta (2,0-10,0 g/min). Como respostas foram avaliadas a distribuição de umidade no leito durante a secagem, teor de umidade do produto, massa de matéria seca retida pelas esferas inertes, eficiência de recuperação do pó, cor do produto em pó e depois de dissolvido em água, propriedades térmicas, teor de proteína, solubilidade em meio aquoso e propriedades reológicas a baixas e altas deformações de géis térmicos de clara de ovo. O uso de planejamento fatorial completo e superfícies de resposta foram fundamentais para avaliar o efeito simultâneo das condições de secagem em leito de jorro fluidizado sobre o processo e o produto obtido. Esta metodologia diminuiu significativamente o número de experimentos e se mostrou eficiente para conhecer a relação de causa e efeito das condições de processo sobre as respostas. Todas as condições de processo estudadas influenciaram nas características do produto obtido, porém a temperatura e a vazão de ar foram as mais importantes na definição da qualidade da clara de ovo seca em leito de jorro fluidizado. Para avaliar a relação entre as várias respostas obtidas e as variáveis de processo, bem como determinar as condições ótimas de operação do secador foi aplicada a técnica estatística da desejabilidade. Concluiu-se que os níveis ótimos de operação do secador seriam: temperatura de 73,7 °C, vazão de ar de 110,8 m³/h, razão vazão de jorro/vazão de ânulo de 1,1 (58,04 m³/h e 52,76 m³/h de vazão de jorro e ânulo, respectivamente) e vazão de pasta de 9,2 g/min. Nestas condições, 60% do total desejado para todas as respostas seria atendido, sendo que a eficiência de recuperação de matéria seca pelo ciclone seria de 74,4% e o grau de desnaturação das proteínas seria baixo (conalbumina: 42,1% e ovalbumina: 25,5%), o que resultaria em elevado grau de solubilidade (em torno de 98%). Neste caso, a temperatura de início de desenvolvimento da estrutura do gel seria estimada em 62 ºC, porém a força da rede seria dada pela desnaturação da ovalbumina (temperatura de desnaturação próxima a 80 oC) e por isto, as características reológicas de géis térmicos de clara de ovo foram estudadas. Os géis térmicos (80 ºC/30 min) de clara de ovo seca em leito de jorro fluidizado mostraram alta deformabilidade e características elásticas similares aos géis obtidos a partir de clara de ovo liofilizada. Assim, a partir da análise dos resultados obtidos foi possível otimizar a secagem da clara de ovo em leito de jorro fluidizado alcançando-se boa eficiência de processo e preservando suas propriedades funcionais / Abstract: The objective of this study was to evaluate an egg white drying process using a two-dimensional spouted fluidized bed dryer with glass spheres as the inert particles. A complete 24 factorial design was used, with 3 central and 8 axial points, using the following independent variables: temperature (55-95ºC), total drying air flow rate (97.1-138.3 m3/h), ratio between spout and annulus air flow rates (0.6-1.4)and paste feed flow rate (2.0-10.0 g/min). As responses, the moisture distribution in the bed during drying, product moisture content, mass of dry matter adhered to inert particles, powder recovery efficiency, product colour as powder and after dissolving in water, thermal properties, protein content, solubility in aqueous medium and rheological properties at high and low deformation of heat-induced egg white gels. The use of a complete factorial design and response surfaces was fundamental to evaluate the simultaneous effect of the drying conditions in a spouted fluidized bed on the process and obtained product. This methodology significantly reduced the number of experiments and showed to be efficient to understand the cause and effect relationship of process conditions on responses. All the process conditions studied influenced the characteristics of the product obtained, but temperature and air flow rate were the most important variables in defining the quality of the egg white dried in a spouted fluidized bed. The statistical technique of desirability was used to evaluate the relationship between the various responses obtained and the process variables, and to determine the optimum dryer operating conditions. It was concluded that the optimum operational levels for the dryer were: temperature of 73.7ºC, air flow rate of 110.8 m3/h, ratio of spout/annulus flow rate of 1.1 (58.04 m3/h and 52.76 m3/h of spout and annulus flow rate, respectively) and paste flow rate of 9.2 g/min. Under these conditions, 60% of the desired total for all the responses would be attained, the recovery efficiency of the dry material by the cyclone would be 74.4% and the degree of protein denaturation would be low (conalbumin: 42.1% and ovalbumin: 25.5%), resulting in very good solubility (about 98%). In this case, the temperature at the start of the gel structure development would be estimated at 62ºC, although the gel strength would be given by the ovalbumin denaturation (denaturation temperature near 80ºC) and for this reason, the rheological characteristics of the thermal egg white gels were investigated. The thermal gels (80ºC/30 min) of egg white dried in a spouted fluidized bed, obtained in this study, showed high deformability and elastic characteristics, similar to the gels obtained from freeze-dried egg white (literature data). Thus, from the analysis of the obtained results it was possible to optimize the drying of egg white in a spouted fluidized bed, obtaining good process efficiency yet preserving the functional properties / Doutorado / Doutor em Engenharia de Alimentos

O aumento na procura por produtos naturais pelo mercado farmacêutico, cosmético e alimentício demanda pesquisas no desenvolvimento desses produtos. Esses são direcionados à obtenção de substâncias de origem vegetal ou animal, assim como, para produtos biotecnológicos. Investigações quanto à atividade antibacteriana de proteínas e peptídeos são realizadas. Dentre essas substancias, podemos citar as lisozimas, proteínas que hidrolisam as ligações β 1-4 glicosídicas entre o ácido N-acetilmurâmico e N-acetilglicosamina, presentes no peptidoglicano da parede celular bacteriana. Além disso, apresentam atividade de quitinase, ou seja, quebram a ligação glicosídica da quitina presente na parede fúngica. As lisozimas apresentam alta especificidade pela parede microbiana indicando aparente ausência de efeitos tóxicos aos humanos. Assim, tornando-a candidata a agente antimicrobiano em formulações cosméticas e farmacêuticas. A lisozima de ovo de galinha tem atividade antimicrobiana, entretanto não havia estudos relacionados com os micro-organismos contaminantes normalmente encontrados em produtos farmacêuticos e cosméticos. Além disso, a lisozima recombinante de Musca domestica (MdL1) não possui ainda sua atividade antimicrobiana definida. Os objetivos do trabalho foram:1) Obtenção da lisozima recombinante de Musca domestica (MdL1); 2) Avaliação a atividade antimicrobiana da MdL1 e de lisozima de ovo de galinha, Hen Egg White Lysozyme (HEWL), frente à Staphylococcus aureus (ATCC 6538), Micrococcus luteus (ATCC 4698), Pseudomonas aeruginosa (ATCC 9027), Escherichia coli (ATCC 8739), Candida albicans (ATCC 10231) e Aspergillus niger (ATCC 16404); 3) Avaliação da toxicidade da lisozima em cultura de células de fibroblastos (ATCC CCL-92). A MdL1 foi obtida por meio de expressão gênica em Pichia pastoris GS115 (Invitrogen), concentrada utilizando polietilenoglicol 6000 e dialisada contra água deionizada através da membrana com porosidade seletiva de 12kDa. A homogeneidade foi analisada por eletroforese em gel de poliacrilamida em condições desnaturantes; e a atividade catalítica foi avaliada utilizando células liofilizadas de Micrococcus luteus como substrato. A atividade antimicrobiana foi determinada por método específico para cada enzima. A toxicidade in vitro das amostras foi avaliada pela viabilidade celular de fibroblastos ATCC CCL-92. A MdL1 obtida apresentou características de homogeneidade adequadas e atividade de 108,35 U/mg. A HEWL mostrou-se ativa contra S. aureus, M. luteus e C. albicans. A MdL1 apresentou-se ativa contra M. luteus, apenas. Devido a ausência de atividade antimicrobiana a MdL1 não foi submetida a avaliação citotóxica. Em relação à HEWL não demonstrou citotoxicidade na avaliação prévia realizada. / The increase in the search for natural products by pharmaceutical, cosmetic and food markets requires researches in these products development. These are directed to obtaining substances of vegetal or animal origin, as well as biotechnological products. The research in relation to antibacterial activity of proteins and peptides is carried out. Among these substances, it is possible to mention the lysozyme, protein that catalyze the break of 1,4-beta-D glucosidic bond between N-acetylmuramic acid and N-acetilglicosamine which are present in peptidoglicane of the bacterial cell wall. Besides, there is kitinase activity, that is, they break the glicosidic bond of chitin which is present in fungal wall. The lysozymes show high specificity by microbial wall indicating apparent absence of toxicological effects to human beings. Therefore, it becomes the candidate to antimicrobial ingredient in cosmetic and pharmaceutical dosage forms. The hen egg white lysozyme has antimicrobial activity, however there were no studies related to spoiled microorganism usually found in pharmaceutical and cosmetic products. In addition, there were not studies about microbial activity of recombinant Musca domestica lysozyme 1 (MdL1). The aim of this research was: 1) To obtain MdL1; 2) Evaluation of MdL1 and hen egg white lysozyme (HEWL) antimicrobial activity against Staphylococcus aureus (ATCC 6538), Micrococcus luteus (ATCC 4698), Pseudomonas aeruginosa (ATCC 9027), Escherichia coli (ATCC 8739), Candida albicans (ATCC 10231) e Aspergillus niger (ATCC 16404); 3) Evaluation of lysozyme toxicity in fibroblast cells culture (ATCC CCL-92). The MdL1 was expressed as recombinant protein in Pichia pastoris GS115 (Invitrogen), concentrated using polyethylene glycol 6000 and dialyzed against deionized water through the selective porosity of 12kDa membrane. The homogeneity was analyzed by electrophoresis in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and the catalytic activity was evaluated using lyophilized cells of Micrococcus luteus as substract. The antimicrobial activity was evaluated using specific methods for each enzyme. The sample toxicity was evaluated by cell viability using ATCC CCL-92 fibroblasts. The MdL1 obtained presented suitable homogeneity characteristics and activity of 108.35 U/mg. The HEWL has showed activity against S. aureus, M. luteus e C. albicans. MdL1 only showed activity against M. luteus. Due to the absence of antimicrobial activity of MdL1 in the evaluated concentration it was not submitted to the cytotoxicity test. Regarding HEWL, it has not showed citotoxicity in the previous test.

Submitted by Johnny Rodrigues (johnnyrodrigues@ufcg.edu.br) on 2018-05-28T17:43:32Z No. of bitstreams: 1 TAMIRES DOS SANTOS PEREIRA - DISSERTAÇÃO PPGSA PROFISSIONAL 2015..pdf: 2702576 bytes, checksum: 45431567404c5290f49012771848f87a (MD5) / Made available in DSpace on 2018-05-28T17:43:32Z (GMT). No. of bitstreams: 1 TAMIRES DOS SANTOS PEREIRA - DISSERTAÇÃO PPGSA PROFISSIONAL 2015..pdf: 2702576 bytes, checksum: 45431567404c5290f49012771848f87a (MD5) Previous issue date: 2015-12-17 / O ovo em sua forma integral ou a clara e gema apresentam-se como ingrediente fundamental em inúmeros produtos alimentares ao agregar propriedades nutricionais e funcionais. A secagem é uma maneira bem sucedida de conservação dos ovos, considerando algumas vantagens como: ocupar menos espaço no estoque; facilidade de transporte; boa uniformidade; utilização mais fácil e ter qualidade microbiológica estável. Uma vantagem considerável na secagem em camada de espuma é a sua capacidade de manter a alta qualidade dos produtos, apresentando grandes possibilidades para a indústria alimentícia, além da baixa temperatura empregada e com tempo de desidratação reduzido devido à maior área de contato com o ar. Este trabalho tem como objetivo estudar a cinética de secagem em camada de espuma das claras dos ovos de granja e de capoeira (caipira), bem como ajustar os dados experimentais das curvas de secagem com modelos cinéticos como o de Henderson e Pabis, Midilli e Kucuk, Page e um modelo linear. Foram determinadas características da espuma, como densidade, expansão, capacidade de incorporação de ar e estabilidade da espuma. O estudo da influência das variáveis independentes sobre a secagem foi feito de acordo com um planejamento experimental fatorial completo 23 + 4 pontos centrais. As variáveis operacionais a serem estudadas (variáveis de entrada) foram: espessura da camada de espuma; tempo de agitação da espuma e temperatura de secagem. E as variáveis dependentes do processo (variáveis de saída) foram tempo de secagem e umidade final, teor de proteínas e luminosidade (cor). Foi realizada a caracterização físico-química, microscópica e de cor do pó obtido. A clara de ovo, por conter naturalmente propriedades espumantes devido ao alto teor e estrutura de suas proteínas apresentou resultados satisfatórios em relação à caracterização física da espuma. Todos os experimentos apresentaram uma boa reprodutibilidade, com curvas representadas caracteristicamente pela taxa constante e a taxa decrescente. Para o ajuste dos dados da clara de capoeira os modelos não lineares que melhor representaram os dados experimentais foram o de Midilli e Kucuk e o modelo de Page seguidos do modelo linear que foi utilizado para o ajuste dos dados até os 150 minutos de secagem. O modelo de Midilli e Kucuk apesar de apresentar o melhor coeficiente de determinação (R2) não foi estatisticamente significativo. Para a clara de granja o modelo linear apresentou o melhor ajuste considerando o tempo máximo de 130 minutos de secagem, seguido pelos modelos não lineares de Midilli e Kucuk e o de Page respectivamente. O processo de secagem da clara de ovo pelo método de camada de espuma mostrou-se satisfatório quanto aos parâmetros avaliados, sendo que as condições testadas não influenciaram de forma significativa em características tecnológicas importantes do produto final, como o teor de proteínas. / The egg in their entirety form or the white and the yolk present as essential ingredient in many food products by adding nutritional and functional properties. Drying is a successful way of conservation of eggs, whereas some advantages as to occupy less space ease of transport, good uniformity, easier to use and be stable microbiological quality. A considerable advantage in the foam-mat drying is its ability to maintain high product quality, presenting great opportunities for the food industry, besides the low temperature employed and reduced dehydration of time due to the larger area of contact with air. This work aims to study the drying kinetics in foam-mat drying the white of the farm eggs and capoeira (caipira) and to adjust the experimental data of the curves of dryness with the kinetic model obtained as Henderson and Pabis, Midilli and Kucuk , Page and a linear model. The study of the influence of the independent variables about dryness was made according to an experimental factorial plan complete 23 + 4 central points. The operational variables to be studied (input variables) were: thickness of the foam layer; shaking time of foam and drying temperature. And the dependent variables of the process (output variables) were drying time and final moisture content, protein content and brightness (color). It was made physicochemical characterization, and microscopic color of the powder obtained was performed. Egg whites to contain naturally foaming properties due to the high content and structure of its proteins showed satisfactory results regarding the physical characteristics of the foam. All experiments showed good reproducibility with curves represented by the characteristically constant rate and decreasing rate. For the adjustment of the data of the capoeira white data nonlinear models that best represent the experimental data were of Midilli and Kucuk and followed by Page linear model was used to fit the data until 150 minutes drying. The model of Midilli and Kucuk despite having the best coefficient of determination (R2) was not statistically significant. For the white of the farm linear model showed the best fit considering the maximum time of 130 minutes drying, followed by non-linear models and Midilli and Kucuk and the Page respectively. The process of drying the egg white froth layer method was satisfactory for the evaluated parameters, and the conditions tested did not influence significantly on important technological characteristics of the final product as the protein.

O aumento na procura por produtos naturais pelo mercado farmacêutico, cosmético e alimentício demanda pesquisas no desenvolvimento desses produtos. Esses são direcionados à obtenção de substâncias de origem vegetal ou animal, assim como, para produtos biotecnológicos. Investigações quanto à atividade antibacteriana de proteínas e peptídeos são realizadas. Dentre essas substancias, podemos citar as lisozimas, proteínas que hidrolisam as ligações β 1-4 glicosídicas entre o ácido N-acetilmurâmico e N-acetilglicosamina, presentes no peptidoglicano da parede celular bacteriana. Além disso, apresentam atividade de quitinase, ou seja, quebram a ligação glicosídica da quitina presente na parede fúngica. As lisozimas apresentam alta especificidade pela parede microbiana indicando aparente ausência de efeitos tóxicos aos humanos. Assim, tornando-a candidata a agente antimicrobiano em formulações cosméticas e farmacêuticas. A lisozima de ovo de galinha tem atividade antimicrobiana, entretanto não havia estudos relacionados com os micro-organismos contaminantes normalmente encontrados em produtos farmacêuticos e cosméticos. Além disso, a lisozima recombinante de Musca domestica (MdL1) não possui ainda sua atividade antimicrobiana definida. Os objetivos do trabalho foram:1) Obtenção da lisozima recombinante de Musca domestica (MdL1); 2) Avaliação a atividade antimicrobiana da MdL1 e de lisozima de ovo de galinha, Hen Egg White Lysozyme (HEWL), frente à Staphylococcus aureus (ATCC 6538), Micrococcus luteus (ATCC 4698), Pseudomonas aeruginosa (ATCC 9027), Escherichia coli (ATCC 8739), Candida albicans (ATCC 10231) e Aspergillus niger (ATCC 16404); 3) Avaliação da toxicidade da lisozima em cultura de células de fibroblastos (ATCC CCL-92). A MdL1 foi obtida por meio de expressão gênica em Pichia pastoris GS115 (Invitrogen), concentrada utilizando polietilenoglicol 6000 e dialisada contra água deionizada através da membrana com porosidade seletiva de 12kDa. A homogeneidade foi analisada por eletroforese em gel de poliacrilamida em condições desnaturantes; e a atividade catalítica foi avaliada utilizando células liofilizadas de Micrococcus luteus como substrato. A atividade antimicrobiana foi determinada por método específico para cada enzima. A toxicidade in vitro das amostras foi avaliada pela viabilidade celular de fibroblastos ATCC CCL-92. A MdL1 obtida apresentou características de homogeneidade adequadas e atividade de 108,35 U/mg. A HEWL mostrou-se ativa contra S. aureus, M. luteus e C. albicans. A MdL1 apresentou-se ativa contra M. luteus, apenas. Devido a ausência de atividade antimicrobiana a MdL1 não foi submetida a avaliação citotóxica. Em relação à HEWL não demonstrou citotoxicidade na avaliação prévia realizada. / The increase in the search for natural products by pharmaceutical, cosmetic and food markets requires researches in these products development. These are directed to obtaining substances of vegetal or animal origin, as well as biotechnological products. The research in relation to antibacterial activity of proteins and peptides is carried out. Among these substances, it is possible to mention the lysozyme, protein that catalyze the break of 1,4-beta-D glucosidic bond between N-acetylmuramic acid and N-acetilglicosamine which are present in peptidoglicane of the bacterial cell wall. Besides, there is kitinase activity, that is, they break the glicosidic bond of chitin which is present in fungal wall. The lysozymes show high specificity by microbial wall indicating apparent absence of toxicological effects to human beings. Therefore, it becomes the candidate to antimicrobial ingredient in cosmetic and pharmaceutical dosage forms. The hen egg white lysozyme has antimicrobial activity, however there were no studies related to spoiled microorganism usually found in pharmaceutical and cosmetic products. In addition, there were not studies about microbial activity of recombinant Musca domestica lysozyme 1 (MdL1). The aim of this research was: 1) To obtain MdL1; 2) Evaluation of MdL1 and hen egg white lysozyme (HEWL) antimicrobial activity against Staphylococcus aureus (ATCC 6538), Micrococcus luteus (ATCC 4698), Pseudomonas aeruginosa (ATCC 9027), Escherichia coli (ATCC 8739), Candida albicans (ATCC 10231) e Aspergillus niger (ATCC 16404); 3) Evaluation of lysozyme toxicity in fibroblast cells culture (ATCC CCL-92). The MdL1 was expressed as recombinant protein in Pichia pastoris GS115 (Invitrogen), concentrated using polyethylene glycol 6000 and dialyzed against deionized water through the selective porosity of 12kDa membrane. The homogeneity was analyzed by electrophoresis in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and the catalytic activity was evaluated using lyophilized cells of Micrococcus luteus as substract. The antimicrobial activity was evaluated using specific methods for each enzyme. The sample toxicity was evaluated by cell viability using ATCC CCL-92 fibroblasts. The MdL1 obtained presented suitable homogeneity characteristics and activity of 108.35 U/mg. The HEWL has showed activity against S. aureus, M. luteus e C. albicans. MdL1 only showed activity against M. luteus. Due to the absence of antimicrobial activity of MdL1 in the evaluated concentration it was not submitted to the cytotoxicity test. Regarding HEWL, it has not showed citotoxicity in the previous test.

The natural ability of filamentous fungi to synthesize, glycosylate, and secrete high levels of protein products has made them potentially attractive hosts for heterologous protein production. Advances in fungal genetics enabled the expression of several high value proteins in filamentous fungi. Particularly the genus, Aspergillus has proven to be potentially useful for the expression of eukaryotic gene products. This thesis pertains to the optimization of recombinant protein production by the fungal host, Aspergillus niger. The target recombinant protein of interest is hen egg white lysozyme (HEWL). This protein encoded in the genome resulting in relatively stable gene construct; however, it is subject to extracellular protease attack. The objective of the proposed research is the development and application of engineering methodology for the analysis and optimization of a fungal bioprocess for recombinant protein production. The underlying hypothesis is that a significant improvement of target protein productivity is achievable by using appropriate optimization techniques. To accomplish this, during the first phase of this study a statistically based experimental method was used to systematically elucidate the effect of medium components (starch, peptone, ammonium sulfate, yeast extract, and CaCl₂.2H₂O) on hen egg white lysozyme production by Aspergillus niger HEWL WT-13-16. A 2⁵⁻¹ fractional factorial design augmented with center points revealed that peptone, starch, and ammonium sulfate were the most significant factors, whereas the other medium components were not important within the levels tested. Then, the method of steepest ascent was employed to approach the proximity of optimum. This task was followed by a central composite design to develop a response surface for medium optimization. The optimum medium composition for lysozyme production was found to be: starch 34 g/L, peptone 34 g/L, ammonium sulfate 11.9 g/L, yeast extract 0.5 g/L, and CaCl₂.2H₂O 0.5 g/L. This medium was projected to produce theoretically 212 mg/L lysozyme. Using this optimized medium, an experimentally observed maximum lysozyme concentration of 209±18 mg/L verified the applied methodology. A second optimization approach was based on metabolic flux analysis (MFA). A comprehensive metabolic network comprising three intracellular compartments (cytoplasm, mitochondrion and peroxisome) was developed for Aspergillus niger. The metabolic flux network included carbohydrate and amino acid metabolism in both anabolic and catabolic reactions. According to experimental observations, the time course of fermentation was divided into five phases, each with unique physiological properties. The network was used to form a set of linear algebraic equations based on the stoichiometry of the reactions by assuming pseudo-steady state for intracellular metabolites. The metabolic flux model consists of 137 metabolites and 287 processes, of which 181 represent biochemical conversions and 106 represent transport processes between the different compartments and the extracellular environment. In addition, due to the physiological evidence some biochemical reactions considered to be active only in one direction. Linear programming was used for optimizing of the specific growth rate as the objective function in combination with 37 measured input and output fluxes of the key metabolites to evaluate corresponding intracellular flux distributions throughout the batch fermentations. The general applicability of the methodology was evaluated by establishing commonality to optimize recombinant HEWL production. The proposed model was able to predict correctly the specific growth rate, oxygen uptake rate, and carbon dioxide evolution rate with good precision. The results of the metabolic flux and sensitivity analysis were employed for medium design. Growth was biphasic; glucose was utilized initially as the carbon source and was followed by its oxidation product, gluconate, later. Logarithmic sensitivity analysis revealed that the addition of proline, alanine and glutamate benefited growth in defined media. The experimental observations and flux analysis showed that tyrosine was a potential candidate for biomass production improvement. The two amino acids, namely proline and tyrosine benefited biomass production during the initial growth phases. Glutamate and alanine were particularly important during the latter stages of the batch process. A series of growth studies were conducted with the identified amino acids added in the medium. In these preliminary nutritional experiments the contribution to growth enhancement was 46% for proline, 23% for glutamate, and 22% for tyrosine. Model predictions were further verified by conducting batch and fed-batch fermentations in a 7- liter bioreactor. The programmed addition of four amino acids (proline, glutamate, alanine, and tyrosine) according to a predetermined schedule resulted in a 44% improvement in biomass and 41% improvement in recombinant protein production. The experiments also confirmed the model prediction that extra amount of amino acids besides the identified ones would not significantly enhance biomass and the recombinant protein production. A computer-based control system was developed for the on-line monitoring and control of the major state variables (e.g., temperature, pH, and DO) during the time course of fermentation. The graphical programming environment, LabVIEW was used to acquire and integrate these variables in a supervisor computer. The temperature of the bioreactor during sterilization and fermentation was controlled using a cascade methodology. The controller parameters of the master and slave loops were determined experimentally to yield a smooth response with minimum overshoot of both the bioreactor and jacket temperatures. The program scheduled various required steps in an established order during the fermentation. This feature of the software guarantees that every necessary operation will be met. The graphical representation of the process is displayed on the screen and helps the user to follow the process and perform the required adjustments. Furthermore, different variables can be observed simultaneously and saved in text or spreadsheet files for further analysis.

The natural ability of filamentous fungi to synthesize, glycosylate, and secrete high levels of protein products has made them potentially attractive hosts for heterologous protein production. Advances in fungal genetics enabled the expression of several high value proteins in filamentous fungi. Particularly the genus, Aspergillus has proven to be potentially useful for the expression of eukaryotic gene products. This thesis pertains to the optimization of recombinant protein production by the fungal host, Aspergillus niger. The target recombinant protein of interest is hen egg white lysozyme (HEWL). This protein encoded in the genome resulting in relatively stable gene construct; however, it is subject to extracellular protease attack. The objective of the proposed research is the development and application of engineering methodology for the analysis and optimization of a fungal bioprocess for recombinant protein production. The underlying hypothesis is that a significant improvement of target protein productivity is achievable by using appropriate optimization techniques. To accomplish this, during the first phase of this study a statistically based experimental method was used to systematically elucidate the effect of medium components (starch, peptone, ammonium sulfate, yeast extract, and CaCl₂.2H₂O) on hen egg white lysozyme production by Aspergillus niger HEWL WT-13-16. A 2⁵⁻¹ fractional factorial design augmented with center points revealed that peptone, starch, and ammonium sulfate were the most significant factors, whereas the other medium components were not important within the levels tested. Then, the method of steepest ascent was employed to approach the proximity of optimum. This task was followed by a central composite design to develop a response surface for medium optimization. The optimum medium composition for lysozyme production was found to be: starch 34 g/L, peptone 34 g/L, ammonium sulfate 11.9 g/L, yeast extract 0.5 g/L, and CaCl₂.2H₂O 0.5 g/L. This medium was projected to produce theoretically 212 mg/L lysozyme. Using this optimized medium, an experimentally observed maximum lysozyme concentration of 209±18 mg/L verified the applied methodology. A second optimization approach was based on metabolic flux analysis (MFA). A comprehensive metabolic network comprising three intracellular compartments (cytoplasm, mitochondrion and peroxisome) was developed for Aspergillus niger. The metabolic flux network included carbohydrate and amino acid metabolism in both anabolic and catabolic reactions. According to experimental observations, the time course of fermentation was divided into five phases, each with unique physiological properties. The network was used to form a set of linear algebraic equations based on the stoichiometry of the reactions by assuming pseudo-steady state for intracellular metabolites. The metabolic flux model consists of 137 metabolites and 287 processes, of which 181 represent biochemical conversions and 106 represent transport processes between the different compartments and the extracellular environment. In addition, due to the physiological evidence some biochemical reactions considered to be active only in one direction. Linear programming was used for optimizing of the specific growth rate as the objective function in combination with 37 measured input and output fluxes of the key metabolites to evaluate corresponding intracellular flux distributions throughout the batch fermentations. The general applicability of the methodology was evaluated by establishing commonality to optimize recombinant HEWL production. The proposed model was able to predict correctly the specific growth rate, oxygen uptake rate, and carbon dioxide evolution rate with good precision. The results of the metabolic flux and sensitivity analysis were employed for medium design. Growth was biphasic; glucose was utilized initially as the carbon source and was followed by its oxidation product, gluconate, later. Logarithmic sensitivity analysis revealed that the addition of proline, alanine and glutamate benefited growth in defined media. The experimental observations and flux analysis showed that tyrosine was a potential candidate for biomass production improvement. The two amino acids, namely proline and tyrosine benefited biomass production during the initial growth phases. Glutamate and alanine were particularly important during the latter stages of the batch process. A series of growth studies were conducted with the identified amino acids added in the medium. In these preliminary nutritional experiments the contribution to growth enhancement was 46% for proline, 23% for glutamate, and 22% for tyrosine. Model predictions were further verified by conducting batch and fed-batch fermentations in a 7- liter bioreactor. The programmed addition of four amino acids (proline, glutamate, alanine, and tyrosine) according to a predetermined schedule resulted in a 44% improvement in biomass and 41% improvement in recombinant protein production. The experiments also confirmed the model prediction that extra amount of amino acids besides the identified ones would not significantly enhance biomass and the recombinant protein production. A computer-based control system was developed for the on-line monitoring and control of the major state variables (e.g., temperature, pH, and DO) during the time course of fermentation. The graphical programming environment, LabVIEW was used to acquire and integrate these variables in a supervisor computer. The temperature of the bioreactor during sterilization and fermentation was controlled using a cascade methodology. The controller parameters of the master and slave loops were determined experimentally to yield a smooth response with minimum overshoot of both the bioreactor and jacket temperatures. The program scheduled various required steps in an established order during the fermentation. This feature of the software guarantees that every necessary operation will be met. The graphical representation of the process is displayed on the screen and helps the user to follow the process and perform the required adjustments. Furthermore, different variables can be observed simultaneously and saved in text or spreadsheet files for further analysis.