Modifying alcalase activity and stability by immobilization onto chitosan aiming at the production of bioactive peptides by hydrolysis of tilapia skin gelatin

The protease from Bacillus licheniformis, commercially known as Alcalase®, was insolubilized and stabilized by immobilization onto activated chitosan. Activation with different agents, such as glutaraldehyde (GLU-Chi), glyoxyl (GLY-Chi) and divinyl sulfone (DVS-Chi) was investigated. The effect of the immobilization protocol, for instance different pH and times, were also evaluated. GLU-Chi showed the highest activity (35.6UNPA/g) with the smallest substrate (N-Boc-l-alanine p-nitrophenyl-ester, NPA), while GLY-Chi showed the highest activity (1.5 UAzocasein/g) using the greatest substrate (azocasein). A 24-h immobilization period was enough to stabilize the enzyme using the three supports under almost all conditions. Operational stability in azocasein hydrolysis was assayed and GLU-Chi showed no activity loss during 5 cycles. DVS-Chi retained around 70 % of its initial activity after the fifth cycle, whereas GLY-Chi activity retained only 10 %. Finally, the biocatalysts were used in the hydrolysis of tilapia skin gelatin aiming the production of peptides with antioxidant activity. The protein hydrolysates obtained using GLU-Chi presented the highest antioxidant activity (36.7 μM Trolox Eq). However, the best results of operational stability were obtained using DVS-Chi, which did not lose its initial activity after 3 consecutive cycles of gelatin hydrolysis.