Aflatoxin B₁ and M₁ Degradation by Lac2 from Pleurotus pulmonarius and Redox Mediators

Abstract

Laccases (LCs) are multicopper oxidases that find application as versatile biocatalysts for the green bioremediation of environmental pollutants and xenobiotics. In this study we elucidate the degrading activity of Lac2 pure enzyme form Pleurotus pulmonarius towards aflatoxin B₁ (AFB₁) and M₁ (AFM₁). LC enzyme was purified using three chromatographic steps and identified as Lac2 through zymogram and LC-MS/MS. The degradation assays were performed in vitro at 25 °C for 72 h in buffer solution. AFB₁ degradation by Lac2 direct oxidation was 23%. Toxin degradation was also investigated in the presence of three redox mediators, (2,2'-azino-bis-[3-ethylbenzothiazoline-6-sulfonic acid]) (ABTS) and two naturally-occurring phenols, acetosyringone (AS) and syringaldehyde (SA). The direct effect of the enzyme and the mediated action of Lac2 with redox mediators univocally proved the correlation between Lac2 activity and aflatoxins degradation. The degradation of AFB₁ was enhanced by the addition of all mediators at 10 mM, with AS being the most effective (90% of degradation). AFM₁ was completely degraded by Lac2 with all mediators at 10 mM. The novelty of this study relies on the identification of a pure enzyme as capable of degrading AFB₁ and, for the first time, AFM₁, and on the evidence that the mechanism of an effective degradation occurs via the mediation of natural phenolic compounds. These results opened new perspective for Lac2 application in the food and feed supply chains as a biotransforming agent of AFB₁ and AFM₁.

Keywords: Pleurotus; aflatoxin B1; aflatoxin M1; biodegradation; laccase; mycotoxins; redox mediators.

Figure 1

Zymography (A) using ABTS as the substrate and SDS PAGE (B and C) of P. pulmonarius LC preparations. NI-not induced; I-induced; D-cell-sample after DEAE cellulose, D-FF-sample after DEAE FF, Sdex-sample after Superdex, M-Marker. The arrows indicate Lac2 bands.

Figure 2

AFB₁ and AFM₁ degradation (%) after three days of incubation at 25 °C, performed by Lac2 and the respective redox mediator in buffered solution (1 mM sodium acetate pH 5). ABTS-[2,2′-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid)]; AS-acetosyringone, SA-syringaldehyde. Values are the mean of three replicates and the error bars represent the standard error measured between independent replicates.

Figure 3

Examples of HPLC chromatograms of AFB₁ (A—negative control, B—positive control, C—sample after degradation by Lac2) and AFM₁ (D—negative control, E—positive control, F—sample after degradation by Lac2).

Figure 3

Examples of HPLC chromatograms of AFB₁ (A—negative control, B—positive control, C—sample after degradation by Lac2) and AFM₁ (D—negative control, E—positive control, F—sample after degradation by Lac2).