Estudo da imobilização de alfa-amilase (Termamyl 2X) na resina Duolite A-568

Abstract

Starch is the second most abundant polysaccharide in the word and it is the main carbohydrate in the human diet. It is distributed in granules, with a semycristalline nature and it is composed of two macromolecules: amylose, an essentially linear polysaccharide, and amylopectin, an extremely branched one. Many of the industrial applications of starch depend on the hydrolysis of this compound and it has been preferentially carried out with the use of amylase enzymes. They can be used free and immobilized, but the immobilized form has several advantages, like the possibility of increasing enzyme activity and stability. In this research, the immobilization process of the enzyme α-amylase, Termamyl® 2X, in ion exchange resin, Duolite® A-568, was studied. First, the following points were evaluated: the activity of free Termamyl® 2X, 0.2 % v/v; it’s stability in relation to pH, for 2 hours, at 0.05 % v/v and preliminary tests to define the best temperature of immobilization by adsorption. Then, a central composite design (CCD) was defined, in which the influence of pH, time and enzyme concentration on the following responses of the immobilization by adsorption were evaluated: immobilized enzyme activity (IA), recovered activity (RA) and efficiency (Ef). From this CCD, the optimal conditions were defined and preliminary tests were carried out in this condition to define the crosslinking procedure with glutaraldehyde to be tested: before or after the addition of the enzyme. After this, a second CCD was carried out, in which the influence of glutaraldehyde’s concentration and the crosslinking time on the responses IA, RA and Ef, in the process of immobilization by crosslinking, were evaluated. The optimization of this planning was also carried out and, in a defined condition (0,2% w/v of glutaraldehyde and crosslinking time of 25 minutes), the activity of the immobilized enzyme, the stability in relation to pH, the storage stability (at 4 °C and pH 6.50, for 25 days), also performed for the optimal condition of the immobilization by adsorption, and the number of reuses were studied. The free enzyme and the immobilized one by crosslinking showed optimum activity at pH 7.00. The free enzyme was more stable at pH 7.00 and 7.50, keeping 62.10% and 60.57% of residual activity, respectively, while the immobilized enzyme showed optimum better stability at pH 7.00, keeping 43,20% of residual activity. In the first CCD, the temperature of 25°C was defined and the optimal condition obtained for the immobilization was as follows: pH 4.82, time of 15 minutes and enzyme concentration of 0.878 % v/v. In the second CCD, it was established that crosslinking should be done prior to the addition of Termamyl® 2X and the optimum condition obtained was: 0.00 % w/v of glutaraldehyde and crosslinking time of 25 minutes. In both immobilization procedures, the validation of the optimal points was successful. When comparing the two CCDs, it was observed that the first one revealed better results: 77.70 U for IA, 30.43 % for RA and 54.84 % for Ef. Immobilization by crosslinking showed the following results: 35.66 U for IA, 31.38 % for RA and 46.51 % for Ef. After 25 days of storage at pH 6.5 and at 4 °C, the enzyme immobilized by adsorption showed a residual activity of 9.57 % and the enzyme immobilized by crosslinking showed 18.23 % of residual activity. Regarding the number of reuses, after three cycles, the enzyme immobilized by crosslinking presented significant residual activity and stability.