Síntese de nanoflores híbridas das lipases de Candida antarctica (CALB) e Thermomyces lanuginosus (TLL)

Abstract

Lipases are among the most used enzymes in biocatalysis. To improve the application of these enzymes, the strategy of enzymatic immobilization is used, which enables the reuse of the catalysts and increases their stability under the reaction conditions. Traditionally, immobilization is performed using supports, however, in some cases there is a decrease in enzyme activity due to unfavorable conformation and diffusion problems. Immobilization of enzymes through structures such as nanoflowers is an innovative, simple and low-cost method when compared to other techniques. In this context, the main objective of this work was to synthesize hybrid biocatalytic nanostructures, similar to flowers, of lipases from Candida antarctica type B (CALB) and from Thermomyces lanuginosus (TLL). The production of nanoflowers occurred by precipitation of lipases with Copper Chloride (CuCl2) and Copper Sulfate (CuSO4) salts. After the synthesis, two conditions were tested: nanoflowers cross-linked with glutaraldehyde and nanoflowers with nanoparticles cross-linked with glutaraldehyde. Glutaraldehyde and nanoparticles were used in order to increase the stability of nanoflowers. The application of a protein quantification method was investigated using Bradford and ultraviolet absorption. Enzyme activity was quantified by hydrolysis of p-nitrophenyl butyrate (pNPB). The catalytic nanostructures were evaluated for catalytic activity, thermal and operational stability under the tested synthesis conditions and characterized by electrophoresis and Fourier- -transform infrared spectroscopy (FTIR). The best result was obtained by producing nanoflowers cross-linked with nanoparticles by CuSO4 precipitation that showed a half-life time of approximately 3 hours and 50 minutes and maintained 70% of activity for 10 cycles and 60% for 5 cycles with CALB and TLL, respectively. By means of electrophoresis, the binding of the nanostructure with glutaraldehyde was observed by difference in the pattern of bands of the free enzyme and the obtained nanoflowers. The immobilization was also analyzed using the FTIR, detecting the presence of the functional groups of the enzymes and the cross-linking agent in the immobilized derivatives. This work showed that it is possible to produce more stable and efficient nanoflowers of lipases using improved protocols, predicting a possible application in several reactions.