Laccase immobilized on metal-polymer composites for wastewater treatment and sensing applications

Abstract

This study explores the multifunctional applications of laccase, a versatile multi-copper enzyme found in fungi, plants, and bacteria. Laccase facilitates the direct reduction of molecular oxygen to water while oxidizing various electron donors, making it appealing for biotechnological applications across sectors such as food, paper and pulp, wastewater treatment, pharmaceuticals, and biosensing. Despite its advantages, challenges related to high costs, instability under harsh conditions of pressure and temperature, and non-reusability in continuous processes necessitate the exploration of enzyme immobilization techniques. In this thesis, laccase enzyme was immobilized on metal-polymer composites comprising either zinc oxide nanoparticles (ZnONPs) or silver-doped ZnONPs (Ag@ZnONPs) embedded in chitosan, polyvinylpolypyrrolidone (PVPP) and polyaniline (PANI) polymers. Characterization studies, including UV-Vis spectroscopy, scanning electron microscopy (SEM), transmission electron microscopy (TEM), and X-ray diffraction (XRD), demonstrate that Ag doping increased the surface area and reduced the bandgap energy without significantly altering nanoparticle size. The resulting polymer composites exhibit high stability and surface area, enabling effective dye adsorption and immobilization matrices for laccase enzyme. The composite beads were studied to remove four dyes: Bismarck brown, orange G, brilliant blue G, and indigo carmine from sewage wastewater and papermill industrial effluent. Applying these composite beads showcases their efficacy with an efficiency of over 90% and 70% chemical oxygen demand (COD) and over 60% and 80% dye removal from wastewater and papermill effluent respectively. Insights from liquid chromatography-mass spectrometry (LC-MS) indicate that laccase facilitated the cleavage of azo bonds and the formation of smaller compounds that were further mineralized by Aspergillus sp. that only thrive on the degradation by-products. Additionally, laccase-activated composites demonstrated significant antibiotic degradation capabilities, removing over 20% more tetracycline and ciprofloxacin when laccase was included, and exhibiting altered degradation pathways with reduced antibiotic activity over time. Finally, the development of a laccase-immobilized ZnO-polyaniline (PANI) nanocomposite biosensor for detecting CTAB highlights the potential for innovative analytical techniques in environmental monitoring. The biosensor exhibited a high sensitivity and wider dynamic linear detection ranges of 0.5 – 100 µM, 200 – 500 µM and 700 – 1900 µM. Overall, the findings underscore the potential of laccase and its immobilization strategies for sustainable biotechnological applications across various domains.