Laccase Immobilization on Carbon-Based Materials Derived from Spent Brewery Grains: Optimization and Stability Evaluation

Enzyme immobilization onto solid supports enhances their stability, reusability, and efficiency. This work investigates the physical immobilization of laccase (Lac) from <i>Trametes versicolor</i> (purchased, EC 1.10.3.2, ≥0.5 U/mg) onto two carbon-based materials: activated carbon (AC) and biochar (BC), obtained from spent brewery grains (SBGs) through microwave pyrolysis (with and without chemical activation, respectively), generating SBG-AC/Lac and SBG-BC/Lac. Various immobilization conditions (pH 3.5-6.5, Lac concentration 1-10 mg/mL) were tested, with immobilization up to 80 ± 6% (for Lac 1 mg/mL, pH 5.0 in SBG-AC/Lac) and maximum activities of 5.5 ± 0.2 U/g (SBG-AC/Lac) and 4.6 ± 0.5 U/g (SBG-BC/Lac) at pH 3.5 and 40 °C. Although SBG-AC led to a higher immobilization %, SBG-BC was a greener alternative, requiring no chemical activation during production. Kinetics analysis with a typical Lac chromogenic substrate revealed higher values of <i>K</i>_M (Michaelis constant) for SBG-BC/Lac compared with free Lac (Lac_f) (indicating lower substrate affinity), but higher stability, retaining ~60% activity after 24 h, while Lac_f was nearly inactive. These results demonstrate the potential of SBG-BC as a sustainable support for Lac immobilization in applications such as wastewater treatment and environmental monitoring.