Produção de ciclodextrinas em reator de leito fluidizado com a enzima ciclodextrina glicosiltransferase imobilizada

• Ano de defesa: 1998
• Autor(a) principal: Tardioli, Paulo Waldir
• Tipo de documento: Dissertação
• Tipo de acesso: Acesso aberto
• Idioma: por
• Instituição de defesa: Universidade Estadual de Maringá; Brasil; Programa de Pós-Graduação em Engenharia Química; UEM; Maringá, PR; Departamento de Engenharia Química
• Palavras-chave em Português: Enzima ciclodextrina glicosiltransferase, CGTase imobilizada, Ciclodextrina glicosiltransferase; Produção; Enzima CGTase; Produção.Ciclodextrinas; Brasil.; Engenharias; Engenharia Química
• Link de acesso: http://repositorio.uem.br:8080/jspui/handle/1/3802

Cyclodextrins (CDs) are cyclic oligosaccharides, formed by a variable number of glucose units, linked to each other by α - 1,4-linkages. The most common are constituted by 6, 7 and 8 glucose units and are denominated α-CD, β-CD and у-CD, respectively. They are produced by the action of the enzyme Cyclodextrin Glycosyltransferase (CGTase) on the starch previously liquefied. Owing to the fact that CDs have an apolar cavity, which favors the encapsulation of a great variety of organic molecules, they have countless applications in the pharmaceutical, food and cosmetics industries, among others. In this work, the main objective was to study the production of CDs in a fluidized bed reactor, as a function of the residence time of the substrate in the bed of particles. CGTase was immobilized into controlled-pore silica (CPS) particles having a mean diameter of the order of 0.42 mm. CGTase originally from Bacillus alkalophilic sp., and cloned in Escherichia coli, was supplied by WACKER, with 193.1 mg of protein/g of liofilized enzyme and specific activity of 63.1 μmols of β-CD/(min.mg of protein), determined at 50°C and pH 8. The enzyme was purified by biospecific affinity chromatography, using Sepharose 6B as support and β-CD as the immobilized ligand. Afterwards, CGTase was immobilized into CPS by covalent bonding, using the silane-glutaraldehyde method. The purification showed a purification factor of 1.2 with an activity recovery of 72%. From the total protein submitted to purification, 94% was recovered, and 65.3% of this total corresponded to the enzyme CGTase. An enzymatic solution with 1.64 mg of protein/mL of solution was obtained with a specific activity of 73.80 μmols of β-CD/(min.mg of protein ). In the immobilization of CGTase, the yield of protein fixation was 28.96 %, producing an immobilized enzyme with 4.66 mg of protein/g of dry support and an activity of 8.62 μmols of β-CD/(min. gEI), which corresponds to an activity yield of 2,53%. The recovery of the total activity offered to immobilization was 28.68%. The production of CDs was made at 50°C, varying the immobilized enzyme load inside the reactor and fixing the porosity of the liquid-solid fluidized bed at 0.5. The substrate used was a solution of dextrin 10 (FLUKA) 100 g/L, with tris-HCl buffer, pH 8, 0.01 M and calcium chloride 5 mM. Samples were collected at the effluent of the reactor and the concentrations of β-CD and у-CD were determined by colorimetric methods using the dyes phenolphthalein and Bromocresol green, respectively. The maximum conversion of the dextrin to CDs, in the fluidized bed reactor, with immobilized CGTase, was about 17%, in which case the residence time was approximately 13 minutes. The production of β-CD was approximately four times superior to that of β-CD, showing that this enzyme is a у-CGTase. The maximum production of β-CD was 11 mM, representing 80% of the total CD produced. With a residence time of approximately 4 minutes, the production of CDs (β-CD and у-CD) was around 15% (10.4 mM of β-CD and 2.3 mM of у-CD). Using a free CGTase, the time necessary to obtain practically the same production of CDs (10.0 mM of β-CD and 2.5 mM of у-CD) is normally 24 hours. This result demontrates the superior quality of the fluidized bed reactor with immobilized enzyme to produce cyclodextrins.