Enhanced thermal stability of Bacillus licheniformis alkaline protease via immobilization on chitosan-based...

Given that current enzyme immobilization strategies often face challenges related to enzyme leaching and activity loss, optimizing novel carrier materials is essential for meeting specific industrial requirements, such as high-temperature stability. This study explores the immobilization of alkaline protease from Bacillus licheniformis on biocompatible and biodegradable chitosan/hydroxyapatite (CS/HA) composites using genipin as a natural cross-linker. Moreover, the composites were synthesized via an in-situ hybridization method and characterized through FTIR, XRD, TGA/DTG, SEM, BET, and Fluorescence spectrometry analyses. Results demonstrated that 30 % of HA content provided an optimal microenvironment, achieving high activity recovery (93.7 ± 1.6 %) with an enzyme loading capacity of 86.2 ± 2.2 %. The immobilized enzyme (BAP@CS/HA 30 %) maintained high catalytic efficiency and remarkable thermal stability, retaining over 60 % of its activity after 5 min at 110 °C, compared to the free enzyme, which has lost more than 99 % of its activity. Additionally, it exhibited enhanced reusability, maintaining 43.1 % of its activity after ten cycles. The immobilized protease also showed improved storage stability, retaining 66.8 ± 1.6 % of its activity after 20 days at 4 °C. Notably, this work underscores the potential of CS/HA composites as safe, eco-friendly carriers for protease immobilization.