Fig. 2: Performance and enzymatic activity stability of laccase assembled on Cellulose-CD-MMT hydrogels.

a Assembled amount (mg g⁻¹) and activity (U g⁻¹) of laccase on Cellulose-CD-MMT hydrogels at different concentrations. The assembled amount of laccase on Cellulose-CD-MMT hydrogels increased with increasing laccase concentration, reaching a maximum loading of 754.5 mg g⁻¹ at 2.0 mg mL⁻¹. Laccase activity sharply declined when the concentration exceeded 2.0 mg mL⁻¹, attributable to the steric hindrance caused by excess enzyme, which altered conformation and hindered substrate binding. To balance enzyme assembly capacity and activity, the Cellulose-CD-MMT hydrogels assembled with laccase at 2.0 mg mL⁻¹ were utilized for the subsequent stability and degradation assays. pH (b), temperature (c), and storage (d) stability of free- and assembled laccase. e Cyclic operation and assembly stability of the assembled laccase. f Lineweaver-Burk plots of free- and assembled laccase. v_max (μmol L⁻¹ min⁻¹) represents the maximum reaction rate of laccase. K_m (mmol L⁻¹) represents the Michaelis constant. A lower K_m means that the assembled enzyme requires a lower substrate concentration to achieve v_max and usually suggests higher affinity for the substrate. A larger value of K_m and v_max indicates lower affinity and higher reaction rate with substrate, respectively. The immobilized enzymes have several inherent shortcomings, including steric hindrance from carriers that may disannul some active sites on the enzymes, as well as the loss of flexibility to bind ligands during degradation. These limitations are directly responsible for the affinity reduction of the immobilized enzymes towards reactant (i.e., higher Michaelis constant: K_m values than free enzymes). g A comprehensive comparison between assembly strategy of laccase on Cellulose-CD-MMT hydrogels in this work and the immobilization methods of laccase on various carriers (such as nano and porous materials) in previous reports in terms of operational range of pH and temperature (maintaining in excess of 80% of enzymatic activity), suitable pH and temperature for environmental remediation, storage stability (14–30 days), and reusability (7 cycles). For (a–f), data are presented as mean ± S.D. from three replicates (n = 3).