Recuperação de lipase BTL-2 de Bacillus thermocatenulatus produzida em Escherichia coli recombinante empregando suportes magnéticos
Lopes, Laiane Antunes
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Magnetic supports have gained attention because the magnetic separation is easy, has low cost and high efficiency. Moreover, it allows the purification and immobilization of a biomolecules in single step. Lipases are hydrolases that catalyze the hydrolysis and the synthesis of esters from glycerol and long chain fatty acids. Lipase from Bacillus thermocatenulatus (BTL-2) has interesting properties such as stability at high temperatures (50 °C), alkaline pH (9.0-11.0) and organic solvent (2-propanol, acetone and methanol). In view of the characteristics that makes BTL-2 a promising lipase for industrial application, concentration and purification is of great importance for implementation in industrial scale. In this context, this study aimed to purify and to immobilize BTL-2 lipase from Bacillus thermocatenulatus produced in recombinant Escherichia coli using magnetic silica microparticles (SMMps). Initially, it was determined the best condition for extraction of the enzyme, changing the pH, buffer and ionic strengh during the sonication. Sodium phosphate buffer 100 mM pH 8.0 has shown to be the best condition for the enzyme extraction. The adsorption of lipase on SMMP-octyl was compared with the adsorption performed using agarose-octyl and silica-octyl. The agarose-octyl support showed the best enzyme adsorption yield (RAE), 92.9%, and higher selectivity in the adsorption of the enzyme. The SMMP-octyl showed higher adsorption capacity per volume of support (189.45 UTBU/mL) than silica-octyl. In order to improve the SMMP adsorption performance, it was changed the octyl groups density on the support surface, by activating with theoretical percentages of 25%, 50% and 75% of octyl groups. SMMP-octyl 75%, after activation with 75% of octyl groups, showed to be the best condition, reaching adsorption with RAE of 92.87% (374.33 UTBU/mL), similar to the obtained using agarose-octyl. The commercial macroporous silica (IM S60P) was also used for comparison of activation procedures with different densities of octyl groups (25, 50, 75 and 100%). The results obtained showed higher activity and the adsorbed protein by support volume with SMMp than silica for all activation degrees. Dessorption tests, however, showed to be possible to dessorve all enzyme from the agarose-octyl, but not from SMMp and silica-octil. The presence of negative charges in silica particles, due to possible ionic interaction with the enzyme may be responsible for this result. The concentration of Triton X-100 0.4% was the best concentration to desorb the enzyme of SMMP-octyl 25%, 75% and 100% and concentration of 0.5% (w/v) was the best concentration for desorption in SMMP-octyl 50%. Adsorption tests in the presence of cellular debris were performed and presented similar behavior those ones observed for the centrifuged and dyalized enzyme solutions: the SMMP-octyl 75% showed higher RAE (71.25%) in the presence of cellular debris. Esterification reactions, using the adsorved enzyme on SMMp in different activation degrees as catalysts, showed that the derivative SMMP-octyl 75% allowed reaching 60.93% conversion in butyrate butyl after 24 hours of reaction. The BTL-2 adsorbed derived directly from cell debris in SMMP-octyl were used in recycles and showed satisfactory conversion in the first cycle, however it decreased in the following cycles.