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Authors
Advisor(s)
Abstract(s)
Nas últimas décadas, a catecol-O-metiltransferase tem sido produzida em
diversos sistemas biológicos usando vectores de expressão, optimizados sobre várias
formulações moleculares. Apesar de todos os organismos descritos na literatura, terem
produzido formas funcionais da enzima, a produtividade máxima encontrada, até ao
momento, foi de 1g de proteína alvo. Actualmente, a investigação reduzida, realizada na
fase upstream do processo produção de SCOMT humana, destaca as produtividades
específicas e volumétricas para vários domínios biofarmacêuticos e neurológicos. Deste
modo, o principal objectivo deste trabalho foi desenvolver um modelo sustentável que
descreva a produtividade e a actividade da hSCOMT baseada nas condições típicas de
fermentação. Baseado num sistema de cultura de Escherichia coli, o presente estudo
destinou-se a determinar a densidade celular máxima e a quantidade activa/óptima de
proteína em erlenmeyers, proporcionando um desempenho importante para o scale-up
do bioreactor.
Inicialmente, estudámos os meios complexo e semi-definido, devido à elevada
produtividade, anteriormente encontrada, tornando o processo mais facilmente
controlado devido ao uso do glicerol, como fonte de carbono principal. Foram
realizados, ensaios em batch, variando parâmetros chave como (temperatura, pH e
agitação) de acordo com o design factorial estabelecido para o desenvolvimento das
experiências. Com base nos resultados obtidos nos ensaios batch, podemos concluir
que, em termos de actividade específica e produtividade da proteína hSCOMT, o meio
complexo reuniu melhores resultados que o meio semi-definido. No entanto,
conseguimos melhorar os valores, em termos de produtividade mássica e volumétrica,
no meio semi-definido, para temperaturas superiores a 30ºC e pH básico. Um modelo
quimiométrico será proposto para simular os dados experimentais baseados nas
condições de operação batch, nomeadamente, pH, temperatura e velocidade de agitação.
In the past decades, recombinant soluble catechol-O-methyltransferase (SCOMT) has been produced in several biological systems using expression vectors, optimized under several molecular formulations. In spite of all organisms described in the literature, produced functional forms of the enzyme, the maximum productivity achieved so far was 1g of target protein. Indeed until now, insignificant research has been conducted in the upstream stage for human SCOMT production, in order to improve specific and volumetric productivities for several biopharmaceutical and neurological domains. Therefore, the main scope of this work was to develop a suitable model that describes the hSCOMT productivity and activity performance on typical fermentation conditions. Based on an Escherichia coli cultivation system, the present study aims for the assay of maximal cell density and optimal active protein amounts in flasks, providing a major milestone for the bioreactor scale-up. Initially, we focus the studies on complex and semi-defined media, due to the higher protein yields previously achieved and more easily process control using glycerol as the main carbon source. Batch tests were carried out, varying the target parameters (temperature, pH and agitation) according to a well established factorial design of experiments. Based on the results obtained we can concluded that, in batch experiments in terms of specific activity and hSCOMT yield, the complex media achieved better results than a semi-defined formulation. Nevertheless, in a semi-defined media at temperatures above 30 ºC and basic pH we can improve activity results and massic or volumetric productivities. A chemometric model will be proposed to simulate the experimental data based on the batch operating conditions, namely temperature, pH and stirring rate.
In the past decades, recombinant soluble catechol-O-methyltransferase (SCOMT) has been produced in several biological systems using expression vectors, optimized under several molecular formulations. In spite of all organisms described in the literature, produced functional forms of the enzyme, the maximum productivity achieved so far was 1g of target protein. Indeed until now, insignificant research has been conducted in the upstream stage for human SCOMT production, in order to improve specific and volumetric productivities for several biopharmaceutical and neurological domains. Therefore, the main scope of this work was to develop a suitable model that describes the hSCOMT productivity and activity performance on typical fermentation conditions. Based on an Escherichia coli cultivation system, the present study aims for the assay of maximal cell density and optimal active protein amounts in flasks, providing a major milestone for the bioreactor scale-up. Initially, we focus the studies on complex and semi-defined media, due to the higher protein yields previously achieved and more easily process control using glycerol as the main carbon source. Batch tests were carried out, varying the target parameters (temperature, pH and agitation) according to a well established factorial design of experiments. Based on the results obtained we can concluded that, in batch experiments in terms of specific activity and hSCOMT yield, the complex media achieved better results than a semi-defined formulation. Nevertheless, in a semi-defined media at temperatures above 30 ºC and basic pH we can improve activity results and massic or volumetric productivities. A chemometric model will be proposed to simulate the experimental data based on the batch operating conditions, namely temperature, pH and stirring rate.
Description
Keywords
Proteína recombinante Proteína recombinante - PH Proteína recombinante - Temperatura Catecol-O-metiltransferase hSCOMT