A heat-resistant intracellular laccase immobilized via cross-linked enzyme aggregate preparation: Characterization,...

Aureobasidium pullulans laccase was immobilized via cross-linked enzyme aggregate (CLEA) and deployed in bisphenol-A (BPA) removal. The immobilization and BPA removal processes were mathematically modeled. The CLEA-treated BPA was evaluated for phytotoxicity. The optimum conditions for CLEA resulting in the highest immobilization yield were ammonium sulfate (60% w/v), glutaraldehyde (30 mM), pH (4.5), time (6 h) and temperature (45 °C). The CLEA retained about 56% of its activity after twelve catalytic cycles. The optimum pH and temperature of the laccase CLEA were 5.5 and 60 °C respectively. The SEM indicated that the laccase CLEA was type II (unstructured). The data obtained from the heat inactivation kinetics and thermodynamic characterization indicated that the CLEA was stable to heat denaturation than the free enzyme. The kinetic parameters obtained for the CLEA with ABTS as substrate were 101.3 µM, 2.94 µmols^-1 mg^-1 and 0.03 dm³ s^-1 mg^-1 for the Km, Kcat and Kcat/Km respectively. The optimum conditions for BPA removal using the CLEA were temperature (30 °C), time (2 h), CLEA (1.0 mg) and BPA concentration (40 mg/L). After the 7th cycle, BPA removal by the laccase CLEA was 63 ± 2.3%. From the germination index values obtained, the CLEA-treated BPA solution showed no phytotoxicity to germinated S. bicolor seeds compared to the untreated (BPA-only) solutions.