Immobilization of chymotrypsin on hierarchical nylon 6,6 nanofiber improves enzyme performance

Immobilized enzymes enable advances in bioprocessing efficiency and bioactive packaging. Enzyme immobilization onto macroscale solid supports is often limited by low protein loading, inadequate access to substrate, and non-ideal orientation to the solid support; immobilization on nanomaterials has improved activity retention, protein loading, and enabled improved performance in extreme environments, yet has practical limitations including handling, recovery. This work describes the immobilization of chymotrypsin to nylon 6,6 in two formats: electrospun nanofibers and planar films. Protein loading, enzyme activity, and kinetics were compared to that of commercially available systems (free chymotrypsin and chymotrypsin immobilized on agarose beads). Electrospun nylon 6,6 nanofibers had an average fiber diameter of 161±73nm, improving protein loading compared to its planar macroscale counterpart. Chymotrypsin immobilized onto nylon nanofibers exhibited shifts in both working optimum pH and temperature with an increase from pH 7.8 to pH 9, and increased optimum temperature by 10°C compared to free enzyme. The nanofibers also enhanced thermostability compared to native enzyme, enzyme on planar films, and the commercial standard agarose beads with 35% activity retained after 12h at 50°C. This work demonstrates the potential of hierarchical nanomaterials in improving enzyme performance, leveraging benefits of both nano and macroscale supports.