Imobilização de lipase de Candida antarctica tipo B em Toyopearl

Abstract

The objective of this work was to study the immobilization of Candida antarctica type B lipase (CalB) by covalent bond using hydrophilic resin named Toyopearl as a support. The influence of activation agents (glycidol, glutaraldehyde and ethylenediamine) in the hydrolytic activity of the biocatalyst was investigated. The enzyme preparations were tested in the hydrolysis of para-nitrophenyl butyrate (PNPB) and in an esterification reaction, butyl butyrate synthesis from butyric acid and butanol. The support was previously characterized by scanning electronic microscopy (SEM), Xray diffraction (DRX) and Fourier transform infra red (FTIR). Superficial area and porosity were evaluated using BET method. Protein concentration and enzymatic activity in the supernatant were determined before and after immobilization process. Best results of hydrolytic activity were obtained using the enzyme immobilized in Toyopearl-Glyoxyl-EDA-Glutaraldehyde (Toyo-GEG), 894.17 ± 43.29 U/g of support, which is 1.56-fold higher than the hydrolytic activity of Novozym 435. The influence of different loadings of protein and the incubation time in the immobilization were also studied. The saturation of support was observed with a load of 40 mg/g of support with 2238.25 ± 27.33 U/g. A decrease in the hydrolytic activity of enzyme preparations was observed for long incubation times. However, thermal stability studies at 60° C, showed that this parameter was important for enzyme stabilization. Thermal stabilization by immobilization was achieved and the immobilized enzyme was more thermal stable than the soluble enzyme. The immobilized lipase prepared at incubation time of 72 hours was 694.59-fold more stable than soluble enzyme and 12.74 -fold than Novozym 435. In organic medium, cycles of synthesis of butyl butyrate was chosen to quantify operational stability. After the seventh cycle, Toyo-GEG retained around 76 % of the initial activity.