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Aflatoxin B 1 and M 1 Degradation by Lac2 from Pleurotus pulmonarius and Redox Mediators

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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.

No MeSH data available.


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.
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toxins-08-00245-f001: 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.

Mentions: Figure 1, panel A shows the zymogram of extracellular LC activity by using ABTS as the substrate; protein bands exhibiting activity in zymogram were also compared with the related electrophoretic pattern stained with Coomassie stain (Figure 1, panel B and C).


Aflatoxin B 1 and M 1 Degradation by Lac2 from Pleurotus pulmonarius and Redox Mediators
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.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5037472&req=5

toxins-08-00245-f001: 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.
Mentions: Figure 1, panel A shows the zymogram of extracellular LC activity by using ABTS as the substrate; protein bands exhibiting activity in zymogram were also compared with the related electrophoretic pattern stained with Coomassie stain (Figure 1, panel B and C).

View Article: PubMed Central - PubMed

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.

No MeSH data available.