Abstract 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.

中文翻译：

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通过固定化到壳聚糖上来改变碱性蛋白酶的活性和稳定性，旨在通过水解罗非鱼皮明胶生产生物活性肽

摘要 来自地衣芽孢杆菌的蛋白酶，商业上称为 Alcalase®，通过固定在活化的壳聚糖上而变得不溶解和稳定。研究了不同试剂的活化，例如戊二醛 (GLU-Chi)、乙醛 (GLY-Chi) 和二乙烯基砜 (DVS-Chi)。还评估了固定方案的效果，例如不同的 pH 值和时间。GLU-Chi 以最小的底物（N-Boc-l-丙氨酸对硝基苯基酯，NPA）显示出最高的活性（35.6UNPA/g），而 GLY-Chi 显示出最高的活性（1.5 UAzocasein/g）最大底物（偶氮酪蛋白）。24 小时的固定时间足以在几乎所有条件下使用三种支持物稳定酶。测定了偶氮酪蛋白水解的操作稳定性，并且 GLU-Chi 在 5 个循环期间没有显示活性损失。在第五个循环后，DVS-Chi 保留了其初始活性的 70% 左右，而 GLY-Chi 活性仅保留了 10%。最后，将生物催化剂用于罗非鱼皮明胶的水解，旨在生产具有抗氧化活性的肽。使用 GLU-Chi 获得的蛋白质水解物具有最高的抗氧化活性（36.7 μM Trolox Eq）。然而，使用 DVS-Chi 获得了最佳的操作稳定性结果，它在连续 3 个明胶水解循环后没有失去其初始活性。使用 GLU-Chi 获得的蛋白质水解物具有最高的抗氧化活性（36.7 μM Trolox Eq）。然而，使用 DVS-Chi 获得了最佳的操作稳定性结果，它在连续 3 个明胶水解循环后没有失去其初始活性。使用 GLU-Chi 获得的蛋白质水解物具有最高的抗氧化活性（36.7 μM Trolox Eq）。然而，使用 DVS-Chi 获得了最佳的操作稳定性结果，它在连续 3 个明胶水解循环后没有失去其初始活性。