Laccase immobilization on a natural lignocellulosic biomass support: optimization, characterization, and bisphenol A...

Laccase (LAC) was immobilized onto sponge gourd (Luffa cylindrica) through a two step method involving physical adsorption followed by glutaraldehyde-mediated crosslinking. The immobilization parameters were systematically optimized, resulting in maximum catalytic activity (52.98 ± 0.85 U/g) and protein loading efficiency (62.09%) under the following conditions: 2.5 mg/L LAC concentration, 15 mg carrier, 90 min adsorption time, 120 min crosslinking time, and 3% glutaraldehyde. Compared to the free LAC, the immobilized LAC showed a 5 °C increase in optimum temperature and a broader pH activity range. It also exhibited significantly improved thermal and pH stability, retaining 33% and 39% more activity after 240 min at 50 °C and 60 °C, respectively. Storage stability was enhanced, with 57% activity retained after 30 days at 4 °C, compared to only 16% for free LAC. Kinetic analysis revealed a moderate increase in K_m (from 0.53 to 0.72 mM) and a decrease in V_max (from 0.85 to 0.69 mmol/L min), suggesting minor diffusional resistance. In Bisphenol A (BPA) degradation tests, the immobilized enzyme achieved complete removal of 15 mg/L BPA within 240 min and maintained 46% removal efficiency after 10 reuse cycles. These findings demonstrate that sponge gourd is a cost effective, biodegradable support material that can significantly enhance LAC performance for potential applications in environmental remediation.