2.3. Enzyme Activities

For enzyme activity measurements, P. chrysosporium was exposed to p-MWCNTs and o-MWCNTs at 1 mg/mL for 3 d, 7 d, 14 d, and 30 d. At designed time points, the fungus balls were filtered out for the filtrate collection by filter paper. The Lac activity was measured using 2,2′-azino-bis(3-ethylbenz-thiazoline-6-sulfonate) (ABTS) as the substrate. Briefly, the filtrate was diluted with the buffer to allow the proper kinetics based on the pre-evaluations. Then, 1.0 mL of diluted filtrate, 0.2 mL of 0.5 mM ABTS, and 2.7 mL of 0.1 M sodium acetate buffer (pH 4.8) were mixed and the absorbance was immediately monitored at 420 nm (UV1600, Shanghai Mapada Instruments Co., Shanghai, China). The initial slope was used for the Lac activity calculation.

For MnP activity assay, the same filtrate was diluted to allow the moderate kinetics according to premeasurements. Then, to 0.4 mL of diluted filtrate was added 0.1 mL of 1.6 mM MnSO₄ and 3.4 mL of 50 mM sodium lactate buffer (pH 4.5). The reaction was triggered by 0.1 mL of 1.6 mM H₂O₂. The absorbance at 240 nm was monitored to obtain the initial slope for MnP activity calculation.