Cerrena unicolor laccases as sustainable biocatalysts for detoxifying hazardous long-chain phthalates and deconstructing plastic mulch films

Widespread use of plastic mulch films (PMFs) in agriculture causes severe contamination with hazardous phthalate esters (PAEs), threatening agroecosystems and crop health. Herein, we investigated two Cerrena unicolor laccases (rLac1 and rLac4) to remediate PAEs and PMFs. Under optimized conditions, rLac1 exhibited reaction rates of 0.27, 0.34, and 0.33 mg/L/h toward dimethyl phthalate (DMP), dibutyl phthalate (DBP), and di-(2-ethylhexyl) phthalate (DEHP), achieving 64.4%, 81.3%, and 79.2% degradation within 24 h, respectively. rLac4 demonstrated comparable activities (0.31, 0.34, and 0.36 mg/L/h) with degradation rates of 74.6%, 81.8%, and 85.8%. These rates rank among the highest reported for laccases. Critically, both laccases showed distinctive substrate preference for long-alkyl-chain homologs (DEHP ≥ DBP > DMP), contrasting with previously characterized laccases that favor short-chain PAEs. This selectivity was elucidated through molecular docking and kinetic analysis, revealing enhanced affinity for long-chain PAE homologs. HPLC-QTOF-MS analysis confirmed a sequential O -dealkylation-like side-chain cleavage mechanism, generating monoester metabolites. Phytotoxicity assays using Brassica chinensis L. as a model crop validated that the enzymatic degradation products exhibited significantly attenuated phytotoxicity compared to parent PAEs, underscoring effective hazard mitigation. Meanwhile, a 30-day enzymatic treatment of polyethylene-based PMFs induced profound depolymerization, evidenced by enhanced surface hydrophilicity, increased carbonyl functionalities, compromised structural integrity, and reduced average molecular weight. These findings demonstrate C. unicolor laccases as sustainable dual-function biocatalysts that are capable of detoxifying hazardous long-chain PAEs and deconstructing PMFs, thereby offering a promising green remediation strategy to mitigate both contaminant hazards in agricultural soils and ensure food chain safety.