Enhanced catalytic performance of Rhizomucor miehei lipase on di-n-butyl and diethylhexyl phthalates: insights into substrate specificity and immobilization strategy

Di-n-butyl (DnBP) and Diethylhexyl Phthalates (DEHP), known as potential endocrine disruptors, are priority pollutants categorized by many regulatory agencies. Enzymatic degradation is a green and efficient approach to remove PEs in the environment. In this study, the DnBP and DEHP degradation performance of Rhizomucor miehei lipase (palatase) in free and immobilized forms on Halloysite nanoclays (HNCs) in an aqueous system was investigated. Upon enzyme immobilization, the alterations in the palatase’s secondary structure were examined using the circular dichroism (CD) analysis. The binding affinity of DnBP and DEHP to palatase was evaluated with molecular docking approaches. The enzyme’s immobilization efficiency and relative activity were found to be 80.3% and 87.8%, respectively. CD results revealed that palatase retained its secondary structure to a significant extent. HNCs-palatase (HNCs-P) exhibited a high stability, as the structural integrity of palatase was mostly preserved. Both free palatase (FP) and HNCs-P fully degraded DnBP and DEHP (100 mg/L) to phthalic acid and a degradation pathway of DnBP and DEHP was suggested. Immobilization prevented the enzyme inhibition caused by the accumulation of metabolites. After seven consecutive uses, HNCs-P was still able to degrade DnBP (63.3%) and DEHP (72.8%). Molecular docking results showed that DEHP had a higher affinity for palatase than DnBP. This study suggests that enzyme immobilization onto HNCs can increase their stability and catalytic performance. FP and HNCs-P effectively hydrolyse ester bonds responsible for phthalate toxicity. Considering their high efficiency, FP and HNCs-P can be used as potential phthalate degraders in various environmental remediation processes.