Zhimin Zhou,Renkuan Li,Tzi Bun Ng,Yunyun Lai,Jie Yang,Xiuyun Ye
Aflatoxin B1(AFB1) is a known toxic human carcinogen and can be detoxified by laccases, which are multicopper oxidases that convert several environmental pollutants and toxins. In this study, a new laccase that could catalyze AFB1degradation was purified and identified from the white-rot fungusCerrena unicolor6884. The laccase was purified using (NH4)2SO4precipitation and anion exchange chromatography, and then identified as Lac 2 through zymogram and UHPLC-MS/MS based on the Illumina transcriptome analysis ofC. unicolor6884. Six putative laccase protein sequences were obtained via functional annotation. Thelac 2cDNA encoding a full-length protein of 512 amino acids was cloned and sequenced to expand the fungus laccase gene library for AFB1detoxification. AFB1degradation by Lac 2 was conducted in vitro at pH 7.0 and 45 °C for 24 h. The half-life of AFB1degradation catalyzed by Lac 2 was 5.16 h. Acetosyringone (AS), Syrinagaldehyde (SA) and [2,2′ -azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid)] (ABTS) at 1 mM concentration seemed to be similar mediators for strongly enhancing AFB1degradation by Lac 2. The product of AFB1degradation catalyzed by Lac 2 was traced and identified to be Aflatoxin Q1(AFQ1) based on mass spectrometry data. These findings are promising for a possible application of Lac 2 as a new aflatoxin oxidase in degrading AFB1present in food and feeds.Keywords:laccase;Cerrena unicolor;aflatoxin B1;biodegradation;detoxification;product;transcriptomeKey Contribution:A product identified to be AFQ1was found from Lac 2-mediated AFB1degradation, indicating that AFB1would be transformed during Lac 2-mediated AFB1degradation.
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.
危险品化学品经营许可证（带存储）