Hidrólise de quitosana a partir de quitosanases e enzimas não-específicas imobilizadas

Abstract

Chitooligosaccharides (COS) are products of the chitosan hydrolysis, which are high value-added because of their biological activities. The chitosan hydrolysis can be catalyzed by acid, although this method requires high temperature and does not have a large selectivity regarding the size of the COS chains, a key feature for the expression of biological activities. Alternatively, the use of enzymes as catalysts for the hydrolysis becomes an interesting option because it requires mild conditions and the enzymes cleave specific bonds. However, processes involving enzymes are quite costly, so it is necessary to implement strategies to reduce the operational costs. Among these strategies, the immobilization of enzymes stands out as a technique that usually increases thermal and pH stability, besides allowing enzyme recycle. The use of non-specific enzymes can be highlighted as another strategy. Thus, the present study investigated the immobilization of cellulase, β-glucosidase, and chitosanases for chitosan hydrolysis. Prior to this study, the chitosanolytic activity of non-specific enzymes was evaluated under different conditions. The highest activity for cellulase was obtained at pH 6.0 , whereas for β-glucosidase, at pH 4.0, both at 55 oC. In order to evaluate the potential of the immobilization techniques, experiments were carried out with adsorption on the cationic resin Streamline SP and covalents bonds on silica-gel microparticles activated with glutaraldehyde. Regarding the adsorption, pH 5.0 favored the immobilization of all enzymes, and in terms of enzyme load, cellulase and quitosanase were favored by the lowest enzymatic load (25 U/g) while β-glucosidase was favored by the highest enzymatic load (50 U/g). When analyzing the covalent bond assays, the enzymes followed the same trend for enzimatic load, while in terms of glutaraldehyde concentration, cellulase and β-glucosidase were favoured by the highest concentration (1.00%), while the chitosanase yielded the best result with the lowest concentration (0.50%). It is noteworthy that thermal stability, pH, and recycle tests were performed to determine the best immobilization strategy for each enzyme. Thus, the best immobilization strategy for cellulase and β-glucosidase was the covalent bonds, compared to the adsorption, since it favored the stability of both enzymes during recycles (conserving 40.00% of the initial activity, compared to 20.00% of adsorption for cellulase, and 14.75% against only 5.75% in the adsorption system for β-glucosidase). Finally, both immobilization strategies succeeded in increasing the stability of the chitosanase, but adsorption showed a marked increase in performance compared to the covalent bond strategy, maintaining 65.00% of the initial activity at the end of the recycles against 44.42% for the covalent system. In conclusion, all three immobilized enzymes were successful in chitosan hydrolysis.