Enhanced activity and operational stability of recombinant acetylxylan esterase from Aspergillus nidulans immobilized...

Acetylxylan esterases are accessory enzymes that hydrolyze ester bonds of acetyl groups in acetylated xylan and belong to the carbohydrate esterase family. This work aims to evaluate the immobilization of a recombinant acetylxylan esterase (AxeAN) from Aspergillus nidulans on various supports to improve its catalytic performance and operational stability. Three agarose-based polymeric supports, a synthesized magnetic nanoparticle, and two commercial matrices were prepared and characterized. Fourier-transform infrared spectroscopy confirmed functional groups introduced by chemical modifications, while X-ray diffraction verified the crystalline magnetite (Fe₃O₄) structure of the nanoparticles. Transmission electron microscopy revealed heterogeneous particle sizes with an average diameter of 166 nm, and magnetic responsiveness was confirmed. AxeAN was successfully immobilized onto the functionalized supports. Immobilization efficiencies above 95% were achieved for amino-functionalized matrices, with recovered activities exceeding 200% for Glyoxyl-agarose-(3-aminopropyl)triethoxysilane nanoparticles (GA-APTES-Nano) and GA-APTES-Nano™ supports. In contrast, Iminodiacetic acid agarose and glyoxyl-agarose displayed low immobilization efficiencies and poor activity retention. Thermal stability assays showed that APTES-Nano and APTES-Nano™ preserved up to 90% residual activity after prolonged incubation at 60 °C, while glutaraldehyde-activated derivatives were less stable. Operational stability revealed that GA-APTES-Nano maintained over 90% activity after six reuse cycles, whereas other derivatives deactivated more rapidly. These results demonstrate the superior performance of amino-functionalized magnetic nanoparticles, particularly GA-APTES-Nano, as robust, reusable platforms for enzyme immobilization, with potential for several scalable biotechnological applications.