Degradation of Aflatoxin B1 and Zearalenone by Bacterial and Fungal Laccases in Presence of Structurally Defined Chemicals and Complex Natural Mediators

Abstract

Aflatoxin B1 (AFB1) and zearalenone (ZEN) exert deleterious effects to human and animal health. In this study, the ability of a CotA laccase from Bacillus subtilis (BsCotA) to degrade these two mycotoxins was first investigated. Among the nine structurally defined chemical compounds, methyl syringate was the most efficient mediator assisting BsCotA to degrade AFB1 (98.0%) and ZEN (100.0%). BsCotA could also use plant extracts, including the Epimedium brevicornu, Cucumis sativus L., Lavandula angustifolia, and Schizonepeta tenuifolia extracts to degrade AFB1 and ZEN. Using hydra and BLYES as indicators, it was demonstrated that the degraded products of AFB1 and ZEN using the laccase/mediator systems were detoxified. Finally, a laccase of fungal origin was also able to degrade AFB1 and ZEN in the presence of the discovered mediators. The findings shed light on the possibility of using laccases and a mediator, particularly a natural plant-derived complex mediator, to simultaneously degrade AFB1 and ZEN contaminants in food and feed.

A New Laccase of Lac 2 from the White Rot Fungus Cerrena unicolor 6884 and Lac 2-Mediated Degradation of Aflatoxin B1

“…Our study showed similar results that buffers at pH 8.0, 9.0 and 10.0 were responsible for AFB 1 degradation without laccase ( Figure 3 D). The extent of AFB 1 degradation catalyzed by Lac 2 at pH 7.0 would be much greater than that in acidic buffers, which was similar to findings on CotA laccase from B. subtilis [ 20 ], but different from T. versicolor laccase with an optimal pH of 4.5. Furthermore, ABTS was studied as the substrate for the stability of Lac 2.…”

“…The open reading frame (ORF) of lac 2 gene encoded a full-length protein of 512 amino acids with an estimated molecular mass of about 60 kDa, which is in the range of molecular masses of most fungal laccases with regular three domains (50-70 kDa) [25]. Reports on laccases responsible for AFB 1 degradation include the following: BsCotA from B. subtilis (Accession: AID81987.1) [20], laccase from T. versicolor (Accession: CAA77015.1 [21], PDB code: 1KYA [26,27]); Lac 2 from P. pulmonarius (Accession: AAX40733.1) [22]; and Ery4 from P. eryngii (Accession: CAO79915.1) [29]. Researchers have been trying to investigate the mechanism by employing both molecular techniques and direct analysis of degradation products.…”

“…The optimal temperature for laccase from T. versicolor was 35 • C [26]. In the case of CotA laccase from B. subtilis, the AFB 1 degradation rate rose when temperature was raised from 20 to 50 • C, but the rate was not reduced when the temperature continued to increase to 80 • C [20]. The optimal temperature for laccase-mediated AFB 1 degradation depends on the source and species of origin of the laccase.…”

“…Enzymatic degradation of AFB 1 is effective and environmentally friendly, especially in food and feed industries [13]. Enzymes reportedly employed for AFB 1 degradation comprise mainly oxidoreductase, including aflatoxin oxidase enzyme (AFO) [14], peroxidase [15], F 420 H 2 -dependent reductases (FDR) [16], Mn peroxidase (MnP) [11,17], myxobacteria aflatoxin degrading enzyme (MADE) [18], bacillus aflatoxin-degrading enzyme (BADE) [19] and laccases [20][21][22]. The mechanisms of AFB 1 degradation for these enzymes have been elucidated since they have different targets on AFB 1 molecules and different active sites: AFO [23,24] and MnP [11] act on the bifuran ring; FDR catalyzes reduction of α, β-unsaturated ester bond [16]; MADE acts on aromatic lactone and the methoxy group of the coumarin ring [18].…”

“…They hypothesized that the deeper modification of the coumarin-like core of the AFB 1 would be responsible for the fluorescence quenching [ 22 ], which was different from the findings of Alberts et al [ 21 ]. In addition, Wang et al investigated whether CotA laccase from Bacillus subtilis ( Bs CotA) was capable of detoxifying AFB 1 and zearalenone (ZEN) using structurally defined chemicals and complex natural mediators, and the degraded products of AFB 1 using the LMS were detoxified [ 20 ]. In more recent studies, Guo et al found that recombinant CotA laccase from Bacillus licheniformis transformed AFB 1 to AFQ 1 and epi-AFQ 1 , which did not suppress cell viability or induce apoptosis [ 30 ].…”