Imobilização e estabilização de tripsina de pâncreas de porco em hidrogel de quitosana e aplicação em reações de hidrólise

Abstract

In this study, trypsin from the porcine pancreas was immobilized on a heterofunctional support prepared by activating chitosan (Chit) hydrogel with glutaraldehyde (GA), then functionalizing it with glycine (Chit–GA–Gly). The catalytic performance of the immobilized trypsin in the hydrolysis reactions was compared with the catalytic performance of the immobilized enzyme on glutaraldehyde-activated chitosan (Chit–GA) and chitosan hydrogel (Chit). The maximum concentration of immobilized protein on Chit–GA–Gly was approximately 16 mg.g

−1 at pH 9.0

(5 mmol.L

−1 buffer sodium carbonate) at 25 ◦C from an offered protein loading of 20 mg.g −1 .

This biocatalyst exhibited maximum specific activity (SA) of 33.1 ± 0.2 nmol.min−1

.mg−1 for benzoyl-DL-arginine-p-nitroanilide (BAPNA) hydrolysis, bigger values when compared to the classic Qui-Glu support. (SA values ranging between 6.7 ± 0.1 nmol.min−1

.mg−1 and 8.1 ± 0.1

nmol.min−1

.mg−1). The Elovich kinetic model was used to describe the adsorption process

using low (3 mg.g

−1) and high (20 mg.g−1) initial protein loadings. The desorption study revealed a 32% desorption rate of trypsin at pH 5.00 and 50 °C, in the presence of Triton-X detergent, highlighting the occurrence of hydrophobic and ionic interactions between trypsin and the support. The optimum temperature for BAPNA hydrolysis catalyzed by the immobilized trypsin (60 ◦C) was 10 ◦C higher than that of its soluble form. Additionally, the immobilized enzyme was 16 to 20 times more stable than its soluble form at 50–55 ◦C. Thermodynamic studies have shown that the stabilization factor of the immobilized enzyme was 20 times greater than the free enzyme at temperatures of 45 and 50°C. Complete hydrolysis of bovine serum albumin (BSA) at 37 ◦C was achieved after 2 h using a soluble enzyme, while for its immobilized form, the hydrolysis yield was 47%. Reuse tests revealed that this biocatalyst retained 37% of its original activity after 10 successive hydrolysis batches. Based on these results, this support could be used as an interesting alternative for producing heterogeneous biocatalysts with high catalytic activity and thermal stability when producing protein hydrolysates.