Obtenção, caracterização e utilização de hidrogel de quitosana e glicerol fosfato para imobilização de lipase de Rhizopus oryzae

Abstract

The concern with the development of less aggressive techniques for the environment contributing to sustainable development, has led to use of enzyme technology and biodegradable materials as an alternative route. Chitosan is a polymer nontoxic, biodegradable and biocompatible obtained from the deacetylation of chitin by-product of the fishing industry. One of the applications of chitosan is as a support for the immobilization of biocatalysts in order to improve certain characteristics, such as stability and reusability. Immobilization may occur for several ways, with no single method that covers every case. In this context, this study investigated the viability of using a chitosan-based hydrogel to immobilization of lipase Rhizopus oryzae (L036P). To do this was made lipase immobilization by physical adsorption and covalent linking of chitosan hydrogel activated with glutaraldehyde. In order to verify significant chemical modifications and the mass loss of samples as a function of temperature, the materials were subjected to thermogravimetric analysis (TG), and infrared spectroscopy with Fourier transform (FTIR). The residual mass was 30% for the free enzyme and 45% for immobilized enzymes and the infrared spectra confirmed the immobilization due to changes in absorption in certain bands. The analysis of the support surface morphology was performed by images obtained by scanning electron microscopy which showed a denser and less porous structure after crosslinking of the hydrogel. The hydrolytic activity of the immobilized lipase was 406.30 U / g for the immobilization by physical adsorption and 439.82 U / g for the immobilization by covalent attachment. Kinetic parameters (km and Vmáx) were determined and there was no difference difference in the amount of km for both immobilized lipase. The Vmáx value has fallen for both immobilizations indicating a possible non competitive inhibition. The thermal stability and storage were evaluated and it was observed an improvement in thermal stability after 150 minutes and the hydrolytic activity of all the materials showed no significant loss after 120 days. The biocatalysts were further characterized as the optimal activity of action as function of temperature and pH using the experimental design technique through rotational composite design with three replications 2² the center point. The results showed that there is a variation in optimal temperature and pH after immobilization being found maximum values of 839.76 U / g for the free lipase (pH 7.5 and 36 °C), 574.18 U / g for lipase immobilized by physical adsorption (pH 7.5 and 50 °C) and 3572.44 U / g to covalently immobilized lipase (pH 8.5 and 60 °C). From the results obtained, it was verified the potential use of hydrogel as lipase immobilization support.