Figure 2.

Screening structurally defined chemical compounds as a mediator of BsCotA in the degradation of Aflatoxin B₁ (AFB₁) and zearalenone (ZEN). (A) Identification of structurally defined chemical compounds as effective mediators. AFB₁ and ZEN (5 μg/mL each) were individually incubated with BsCotA (0.03 U/mL) and one of the mediators (1 mM) in 50 mM Tris–HCl buffer (pH 7.0) at 30 °C for 10 h. The compounds used were: 1, no compound control; 2, p-coumaric acid; 3, syringic acid; 4, vanillin; 5, syringaldehyde; 6, caffeic acid; 7, 1-hydroxybenzotriazole (HBT); 8. gallic acid; 9, vanillic acid; 10, methyl syringate. (B) Time-course analysis of AFB₁ and ZEN transformation by BsCotA in the presence of methyl syringate. AFB₁ and ZEN (5 μg/mL each) were individually incubated with BsCotA (0.03 U/mL) and 1 mM of methyl syringate in 50 mM Tris–HCl buffer (pH 7.0) at 30 °C for 10 h. The samples were periodically taken for HPLC analysis. Effects of (C) temperature and (D) pH on AFB₁ and ZEN transformation by BsCotA in the presence of methyl syringate. For (C), AFB₁ and ZEN (5 μg/mL each) were individually incubated with BsCotA (0.03 U/mL) and 1 mM of methyl syringate in 50 mM Tris–HCl buffer (pH 7.0) at a temperature ranging from 20 to 80 °C for 10 h. For (D), the incubation was carried out in a buffer with the pH ranging from 2.0 to 12.0. Impacts of (E) metal ions on AFB₁ and (F) ZEN transformation. AFB₁ and ZEN (5 μg/mL each) were individually incubated with BsCotA (0.03 U/mL) and 1 mM of methyl syringate in 50 mM Tris–HCl buffer (pH 7.0) and one of the metal ions (10 mM) at 30 °C for 10 h. Each assay was carried out with three independent biological replicates.