Imobilização direcionada e reversível da L-arabinose isomerase de Enterococcus faecium BFIQ E36 em agarose através do domínio lectina β-trebol (LSLt)

• Ano de defesa: 2018
• Autor(a) principal: Felix, Anne Kamilly Nogueira
• Orientador(a): Gonçalves, Luciana Rocha Barros
• Tipo de documento: Tese
• Tipo de acesso: Acesso aberto
• Idioma: por
• Palavras-chave em Português: L-arabinose isomerase; LSLt; Lectina; Cristalização; Imobilização direcionada
• Link de acesso: http://www.repositorio.ufc.br/handle/riufc/63506

D-tagatose, a type of rare sugar that has 92% sweetness when compared to sucrose, but with 1/3 of its energy value, is obtained from the isomerization of D-galactose by the enzyme L arabinose isomerase EC 5.3.1.4. This high value-added enzyme obtained from Enterococcus faecium had its three-dimensional structure predicted from crystallographic studies and structural modeling, making it possible to design a recombinant enzyme with the insertion of a fusion label, LSLt (lectin Laetiporus sulphureus), which allows the enzyme to be efficiently, simply and inexpensively purified in a single affinity step, having carrier agarose and lactose as eluent. A TEV endoprotease recognition sequence (Tobacco Etch Virus) was also inserted, allowing cleavage of the fusion label following a purification process. The success in the construction resulted in an active enzyme and of easy purification, since the support does not require any type of activation. In the characterization of the enzyme, 50°C and 5.5 were obtained as the ideal temperature and pH, respectively. The studies also showed that the presence of the Mn2+ ion positively affects the catalytic activity of LSLt-LAI. As for immobilization, which occurred after 15 minutes, a yield of 95% was obtained, with activity recovered around 88%. The study of D-galactose bioconversion in D-tagatose showed promising results, 28%, high conversion rate when compared to studies already performed with the enzyme obtained from Enterococcus faecium in its native or recombinant form.