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Utilização de enzima imobilizada para a produção de biodiesel
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Abstract(s)
Procedeu-se à imobilização da cutinase recombinante de Escherichia coli (provenientes de diferentes lotes M3, J1 e J2) por adsorção em suportes hidrofóbicos (Amberlite XAD-2, XAD-4, XAD-7, XAD-1180, Chauny XAD-16 e DIAION HP20), suportes inorgânicos (zeólitos NaA, NaX, CaA, NaY e alumina), suportes iónicos (Dowex 50W-X8, AG 50W-X8, AG 50W-X2, Amberlite IRC 50, IRA-400, IRA-458) e por ligação covalente (nos suportes comerciais metacrilato de epóxi butilo, metacrilato de epóxi e metacrilato de amina). O sobrenadante enzimático em contato com os suportes foi caraterizado a nível da atividade estereolítica (p-NPB) e proteína total (método de bradford). Estudou-se a capacidade de esterificação da cutinase imobilizada nos diferentes suportes na síntese do laureato de propilo e do caproato de hexilo. Por último, foi estudada a reação de transesterificação para a produção do biodiesel utilizando suportes selecionados a partir dos estudos anteriores.
Os suportes hidrofóbicos (Amberlite XAD-7 e Amberlite XAD-1180), suporte inorgânico (alumina), suportes iónicos com ajuste de pH (Dowex 50W-X8, AG 50W-X8 e AG 50W-X2) e suportes por ligação covalente (metacrilato de epóxi butilo) apresentaram uma diminuição mais acentuada dos valores de atividade no sobrenadante e na proteína presente, o que representa maior quantidade de enzima imobilizada nos respectivos suportes. A cutinase (M3) imobilizada no suporte inorgânico alumina apresentou a melhor conversão (63%) na reação de síntese de caproato de hexil em comparação com os suportes hidrofóbicos e iónicos, mas menor do que Cal B (89%). Com os estudos da enzima imobilizada (M3 Concentrada) na alumina pré-equilibrada com a atividade de água a 0,97, pode-se concluir que sem a adição de água no início da reação de síntese do caproato de hexilo esta apresenta uma conversão de 83,3%. A cutinase (J2) imobilizada no suporte covalente metacrilato de epóxi butilo apresentou um grau de conversão (cerca de 90%) constante ao fim de 29 reutilizações. Na síntese do biodiesel, a partir da transesterificação do trioleína e metanol, a Cal B apresentou síntese de alguns ésteres e monoglicerídeos com a adição estereoquímica de metanol por 3 vezes ao longo do tempo da reação em relação à adição de metanol todo no início da reação. No entanto, a CAL B não realiza transesterificação com butanol e etanol adicionados no início da reação. Em oposição, a cutinase do lote J2 imobilizada por ligação covalente no suporte metacrilato de epóxi butilo mostra capacidade de síntese de alguns ésteres e monoglicerídeos quando a reação é feita com a adição de etanol no início da reação e sem adição de água no início da reação.
Recombinant cutinase from Escherichia coli (from diferent batches M3, J1 and J2) was immobilized on hydrophobic supports (Amberlite XAD-2, XAD-4, XAD-7, XAD-1180, Chauny XAD-16 and DIAION HP20), inorganic supports (zeolites NaA, CaA, NaY, NaX and alumina), ionic supports (Dowex 50W-X8, AG 50W-X8, AG 50W-X2, Amberlite IRC 50, IRA-400, IRA-458) and throught covalent binding (on commercial supports epoxy/butyl methacrylate, epoxy metacrylate and amino methacrylate). The supernatant enzyme in contact with the supports was characterized by stereolytic activity (p-NPB) and total protein (bradford method). The esterification ability of immobilized cutinase on the different supports in the synthesis of propyl laureate and hexyl caproate was studied. At last, the transesterification reaction for biodiesel production was studied using selected supports from previous studies. The hydrophobic supports (Amberlite XAD-7 and Amberlite XAD-1180), inorganic support (alumina), ionic supports with pH adjustment (Dowex 50W-X8, AG 50W-X8 and AG 50W-X2) and covalent binding supports (epoxy/butyl methacrylate) showed a more visible decrease in activity and protein values, which represents a greater amount of enzyme immobilized in the respective supports. Immobilized cutinase (M3) on inorganic support alumina showed the best conversion (63%) of hexyl caproate in esterification reaction compared to hydrophobic and ionic supports, but less than Cal B (89%). With the studies of immobilized enzyme (M3 concentrated) in alumina pre equilibrated with water activity at 0.97, it can be concluded that without the adition of water at the beginning of the synthesis reaction of hexyl caproate has a conversion of 83.3%. The cutinase (J2) immobilized on the covalent epoxy/butyl methacrylate support presented a constant degree of conversion (around 90%) at the end of 29 reuses.In the synthesis of biodiesel from the transterification of triolein and methanol, Cal B synthesized some esters and monoglycerides with the stereochemical addition of methanol 3 times during the reaction time in relation to the addition of all the methanol at the beginning of the reaction. However, CAL B does not transesterify with butanol and ethanol added at the start of the reaction. In contrast, cutinase from batch J2 immobilized by covalent binding on the epoxy/butyl methacrylate support shows the ability to synthesize some esters and monoglycerides when the reaction is carried out with the addition of ethanol at the beginning of the reaction and without the addition of water at the beginning of the reaction.
Recombinant cutinase from Escherichia coli (from diferent batches M3, J1 and J2) was immobilized on hydrophobic supports (Amberlite XAD-2, XAD-4, XAD-7, XAD-1180, Chauny XAD-16 and DIAION HP20), inorganic supports (zeolites NaA, CaA, NaY, NaX and alumina), ionic supports (Dowex 50W-X8, AG 50W-X8, AG 50W-X2, Amberlite IRC 50, IRA-400, IRA-458) and throught covalent binding (on commercial supports epoxy/butyl methacrylate, epoxy metacrylate and amino methacrylate). The supernatant enzyme in contact with the supports was characterized by stereolytic activity (p-NPB) and total protein (bradford method). The esterification ability of immobilized cutinase on the different supports in the synthesis of propyl laureate and hexyl caproate was studied. At last, the transesterification reaction for biodiesel production was studied using selected supports from previous studies. The hydrophobic supports (Amberlite XAD-7 and Amberlite XAD-1180), inorganic support (alumina), ionic supports with pH adjustment (Dowex 50W-X8, AG 50W-X8 and AG 50W-X2) and covalent binding supports (epoxy/butyl methacrylate) showed a more visible decrease in activity and protein values, which represents a greater amount of enzyme immobilized in the respective supports. Immobilized cutinase (M3) on inorganic support alumina showed the best conversion (63%) of hexyl caproate in esterification reaction compared to hydrophobic and ionic supports, but less than Cal B (89%). With the studies of immobilized enzyme (M3 concentrated) in alumina pre equilibrated with water activity at 0.97, it can be concluded that without the adition of water at the beginning of the synthesis reaction of hexyl caproate has a conversion of 83.3%. The cutinase (J2) immobilized on the covalent epoxy/butyl methacrylate support presented a constant degree of conversion (around 90%) at the end of 29 reuses.In the synthesis of biodiesel from the transterification of triolein and methanol, Cal B synthesized some esters and monoglycerides with the stereochemical addition of methanol 3 times during the reaction time in relation to the addition of all the methanol at the beginning of the reaction. However, CAL B does not transesterify with butanol and ethanol added at the start of the reaction. In contrast, cutinase from batch J2 immobilized by covalent binding on the epoxy/butyl methacrylate support shows the ability to synthesize some esters and monoglycerides when the reaction is carried out with the addition of ethanol at the beginning of the reaction and without the addition of water at the beginning of the reaction.
Description
Relatório de Dissertação do Mestrado em Engenharia Biológica e Química
Keywords
Cutinase Imobilização de enzimas Transesterificação Esterificação Biodiesel Supports Enzyme immobilization Transesterification Esterification Biodiesel
Citation
Publisher
Instituto Politécnico de Setúbal. Escola Superior de Tecnologia do Barreiro