Avaliação de estratégias de imobilização e estabilização de β-galactosidase de Bacillus licheniformis

Abstract

The dairy industry has expressive participation in the food sector, with the production of dairy products with lactose content reduced by the enzyme β-galactosidase, in increasing demand. Lactose-free products have a higher production cost than dairy products with lactose due to the enzymatic hydrolysis step using soluble enzymes, so the use of immobilized β- galactosidase emerge as an alternative to the high cost of enzymes in their free form Researchesare continuously directed to improve the biocatalyst, using supports and conditions that make the final process feasible and improved. At the first moment in the research, an exploratory study was carried out on the effect caused by ionic strength and pH in the process of immobilization by physical adsorption on Duolite A 568 support. High concentrations of the Britton-Robinson (BR) buffer solution were used, from 300 mM to 1000 mM, varying the pH between 3.5 to 4.5, keeping the immobilization time at 2 hours. During the experiments, the unexpected appearance of enzymatic clusters (granules) was observed, which showed relevant enzymatic activity of 719 (U) at pH 4.0 (300 mM), 767 U at pH 4.5 (300 mM) and 348 U at pH 4.0 (500 mM), indicating possible support-free immobilization. For high buffer molarity conditions (1000 mM), the results showed no improvement in the activity of the immobilized enzyme. In sequence, the joint influence of ionic strength and immobilization time in a central rotational composite design (PCCR) was evaluated together with the analysis of the immobilization process by the yield, efficiency and recovery responses. For the hydrolytic activity of the β-galactosidase enzyme, the best results were between 396 U to 470 U, which were concentrated in short or long periods associated with low ionic strength conditions, evidencing the interaction effect in the studied ranges between the BR buffer concentration (5. 12 to 99.88) and time (0.46 to 6.12 hours).In the parameters of the effect of the analysis of the immobilization performance in observation the highest activity of the enzyme of 470 U under conditions of 86 mM in 5.3h one of the best regions was obtained 91.01 of yield, 96.71 of efficiency and 88, 01 recovery proving to be a good approach to deepening in new research. This research demonstrated that the concentration of the ionic strength of the buffer with defined times and pH of the composition of the medium are important parameters that affect the catalytic active site of the enzyme in its stability, which can benefit or inhibit the activity of the immobilized enzyme.