Laccase immobilization in zeolitic imidazolate metal-organic frameworks for water micropollutant removal via biocatalysis and adsorption

The increasing presence of micropollutants in aquatic environments, originating from diverse industrial sources, poses severe risks to human health, animal life, and ecosystems, even at trace concentrations. Enzyme-based biocatalysis offers a promising green strategy for mitigating these contaminants due to enzymes’ high selectivity and ability to catalyze specific reactions without generating harmful by-products. However, the practical application of free enzymes is often limited by their instability under environmental stressors such as extreme pH and temperature, which leads to denaturation and reduced catalytic performance. To address these challenges, this study investigates the in-situ encapsulation of laccase from Trametes versicolor within metal–organic frameworks (MOFs), specifically Zeolitic Imidazolate Frameworks (ZIF-8, ZIF-67, and ZIF-7). Aqueous-based synthesis was employed, replacing conventional organic solvents with water, thereby emphasizing a green and sustainable approach to MOF fabrication. The crystallization of ZIFs followed a common pathway involving rapid coordination of metal cations (Zn²⁺ or Co²⁺) with imidazolate linkers, nucleation, and subsequent crystal growth governed by metal-to-ligand ratios. This process enabled simultaneous enzyme encapsulation during framework formation, yielding L@ZIF composites. Characterization using FESEM, XRD, FTIR, TGA, BET, and particle size analysis confirmed distinct morphologies, with truncated rhombic dodecahedra in ZIF-8 (1:16), leafy and rod-like crystals in ZIF-67 (1:8 and 1:12), and amorphous spheres in ZIF-7 across all ratios. Laccase loading efficiencies were 65.44% for L@ZIF-8, 81.01% for L@ZIF-67, and 70.74% for L@ZIF-7, where high loading is crucial for maximizing catalytic activity and material efficiency. Enzyme kinetics of free laccase, determined via Hanes-Woolf plot using ABTS as substrate, showed Km = 0.27 mM and Vmax = 0.0031 mM/min, indicating slight low substrate affinity and catalytic turnover to the relative literature, serving as benchmarks to evaluate the performance of the immobilized counterparts. The adsorption rate for ZIF-8 and ZIF-67 was typically faster at the beginning of the adsorption process and started to level off after 240 min (58.1% for ZIF-8 and 6.3% for ZIF-67). The sorption kinetic data for both ZIF-8 and ZIF-67 was observed fitted to the pseudo-first-order kinetics model (R2 > 0.91). ZIF-8 obeyed the Langmuir isotherm model adsorption behaviour, while ZIF-67 Freundlich followed model. These results indicates that ZIF-8 go through monolayer adsorption in homogenous surface while ZIF-67 undergoes multilayer adsorption. Biocatalytic performance tests revealed that L@ZIF-8 achieved over 90% methylene blue degradation within four weeks, surpassing other composites. L@ZIF-8 have higher methylene blue removal compared to its pristine; 92.31% and 85.81% respectively after 4 weeks. Meanwhile, L@ZIF-67 have lower methylene blue removal of 48.78% compared to pristine of 53.36% due to enzyme high confinement that reduce the enzyme reaction. This study establishes a sustainable synthesis route for enzyme-MOF composites using water as a green solvent, avoiding hazardous organic systems. By combining high enzyme loading more than 60% and preserved catalytic activity up to 92% when encapsulate in ZIF, the findings demonstrate the potential of aqueous-synthesized enzyme-MOF composites as eco-friendly and efficient materials for micropollutant remediation, contributing to global sustainability goals such as SDG 6 (Clean Water and Sanitation) and SDG 12 (Responsible Consumption and Production).