PEI-Treated Eggshell and Onion Membranes as Natural Supports for Urease Immobilization: A Performance and Stability Evaluation

Jack Bean urease (JBU) has considerable biotechnological value, but its free form suffers from low stability and is expensive to recover. Immobilization on sustainable, waste-derived supports such as eggshell membrane (ESM) and onion membrane (OM) offers a promising alternative. For the first time, this study provides a systematic and comparative evaluation of JBU immobilization on PEI-pretreated ESM and OM. The systems were assessed in terms of morphology, enzyme kinetics, stability, and resistance to heavy metals after optimization. SEM analysis showed ESM’s fibrous structure allowed for greater enzyme loading than OM. Immobilization increased the apparent K_m (∼1.7-fold for ESM, ∼5.3-fold for OM), suggesting diffusion limitations, while the apparent V_max also increased, following the trend OM > ESM > free JBU, which is likely due to the high local enzyme concentration on the support surface. OM-immobilized JBU exhibited better thermal (stable up to 80 °C) and dry storage stability, whereas ESM-JBU showed superior reusability (50% activity retained after 4 vs 2 cycles). Both systems demonstrated remarkable protection against Cu²⁺ inhibition; at concentrations that fully inactivated the free enzyme, the immobilized systems retained over 70% of their activity. Overall, a clear performance trade-off emerges, with ESM excelling in enzyme loading and reuse, while OM favors operational stability. This comparative analysis highlights the potential of these waste-derived biomaterials as tunable, cost-effective platforms for developing robust immobilized enzyme systems for various biotechnological applications.