Application of carboxymethyl cellulose-g-poly(acrylic acid-co-acrylamide) hydrogel sponges for improvement of efficiency, reusability and thermal stability of a recombinant xylanase

Enzyme immobilization onto supports could improve its specificity, storage stability, reusability, pH tolerance and thermal stability. Aiming to provide a proficient immobilization carrier for a recombinant xylanase (PersiXyn1), the high-performance 3D copolymeric network of carboxymethyl cellulose (CMC)-based hydrogel sponges were synthesized using in situ graft radical polymerization. While typical purified gel drying in a vacuum oven made CMC/Acrylate-Hydrogel, the freeze-drying of the resultant swelled Hydrogel containing different amounts of water, resulted in two samples of sponges (i.e., CMC/Acrylate-Sponge1 and CMC/Acrylate-Sponge2). The structural characteristics and water absorbency properties of all the as-prepared samples were evaluated. CMC/Acrylate-Sponge2 was selected for PersiXyn1 immobilization due to its homogeneous porous polymeric skeleton and maximum water absorbency (166.6 g/g) offering superb enzyme immobilization efficiency (100%). Moreover, presence of numerous oxygenated functionalities and cage-like characteristics in the CMC/Acrylate-Sponge2, led to effective electrostatic attractions between support and enzyme providing physically stabilized and reusable bio-conjugates which exhibited up to 60% recovered activity even after eight continuous reuse cycles. The results of kinetic studies indicated that the specific activities of free and immobilized PersiXyn1 were 4157 and 5508 µmol min⁻¹ mg⁻¹, respectively. Interestingly, CMC/Acrylate-Sponge2 support could noticeably broaden the temperature endurance ranges of immobilized PersiXyn1. While free enzyme could preserve only 10% of its maximum activity, the immobilized enzyme maintained 35% of its maximum activity after 60 min incubation at 60 °C. Lastly, CMC/Acrylate-Sponge2 support provided remarkable enzyme protection from denaturing, which caused shifting enzyme unfolding temperature from 71.5 to 78.0 °C, indicating the effectual shielding effects of polymeric support on PersiXyn1.