Imobilização da lipase de Thermomyces lanuginosus em resina de interação hidrofóbica Streamline™ phenyl

Abstract

Lipases are among the most used enzymes at industrial levels due to their ability to catalyze different reactions and maintain high stability and activity under several reaction conditions. Mainly in the industrial area, these enzymes should be used in their immobilized form since the immobilized catalyst exhibits several advantages compared to its free form. The immobilization process allows the reuse of the enzyme in multiple reaction cycles, reducing processing cost and recovering the enzyme after reaction, avoiding contamination of the final product. Thus, this work seeks to establish an efficient protocol for immobilization of Thermomyces lanuginosus lipase in a hydrophobic resin, Streamline phenyl, producing stable and active derivatives (iTLL) for commercial application. For this, four types of biocatalysts were prepared using, or not, glutaraldehyde (GLU) as a crosslinking agent and polyethyleneimine (PEI) to stabilize the immobilized enzymes. The immobilized biocatalysts, iTLL, iTLL-GLU, iTLL-PEI, and iTLL-PEI-GLU were evaluated according to some immobilization parameters, such as recovered activity to the free enzyme, thermal stability at 65 °C, and operational stability in consecutive cycles of pNPB hydrolysis. Furthermore, the desorption of the enzyme from the support was evaluated using the surfactant cetyltrimethylammonium bromide (CTAB) 0.6%. Increasing the protein load on the immobilization parameters was also investigated. The results showed that immobilization via hydrophobic interaction with the resinproduced biocatalysts (iTLL) with recovered activity values above 100%, with the highest value reaching 106%. The immobilized biocatalysts had advantages and disadvantages compared to enzymatic activity. Still, the iTLL-PEI-GLU derivative was the most stable among the others, despite losing little activity compared to iTLL, presenting the highest thermal stability, with a half-life time of 4h and 27 minutes and the best operational stability retaining 90% of its relative activity after the 5th cycle of pNPB hydrolysis. Furthermore, the enzyme desorption rate was less than 30% using CTAB surfactant for the iTLL and iTLL-GLU. Finally, the increase in enzyme load during immobilization rendered a proportional increase in theoretical activity and biocatalyst activities without influencing the immobilization yield and the recovered activity. Thus, this work showed that the immobilization of lipase from Thermomyces lanuginosus via hydrophobic interaction was efficient, generating derivatives that could be used in industrial processes