Immobilization of enzymes on functionalized cellulose nanofibrils for bioremediation of antibiotics: Degradation mechanism, kinetics, and thermodynamic study

The deteriorating environmental conditions due to increasing emerging recalcitrant pollutants raised a severe concern for its remediation. In this study, we have reported antibiotic degradation using free and immobilized HRP. The functionalized cellulose support was utilized for efficient immobilization of HRP. Approximately 13.32 ± 0.52 mg/g enzyme loading was achieved with >99% immobilization efficiency. The higher percentage of immobilization is attributed to the higher surface area and carboxylic groups on the support. The kinetic parameter of immobilized enzymes was Km = 2.99 mM/L for CNF-CA@HRP, which is 3.5-fold more than the Michaelis constant (Km = 0.84794 mM/L) for free HRP. The Vmax of CNF-CA@HRP bioconjugate was 2.36072 mM/min and 0.558254 mM/min for free HRP. The highest degradation of 50, 54.3, and 97% were achieved with enzymatic, sonolysis, and sono-enzymatic with CNF-CA@HRP bioconjugate, respectively. The reaction kinetics analysis revealed that applying ultrasound with an enzymatic process could enhance the reaction rate by 2.7–8.4 times compared to the conventional enzymatic process. Also, ultrasound changes the reaction from diffusion mode to the kinetic regime with a more oriented and fruitful collision between the molecules. The thermodynamic analysis suggested that the system was endothermic and spontaneous. While LC-MS analysis and OTC's degradation mechanism suggest, it mainly involves hydroxylation, secondary alcohol oxidation, dehydration, and decarbonylation. Additionally, the toxicity test confirmed that the sono-enzymatic process helps toward achieving complete mineralization. Further, the reusability of bioconjugate shows that immobilized enzymes are more efficient than the free enzyme.