Characterisation of ZPH13 phosphotransferase from Bacillus subtilis in zearalenone transformation

Zearalenone (ZEN), a mycotoxin produced by Fusarium species, poses considerable health risks to humans and animals because of its residues in maize and its industrial byproducts. Effective methods to control or eliminate ZEN are urgently needed. Herein, Bacillus subtilis strain Z1Y3, capable of efficient ZEN transformation, was isolated from ZEN-contaminated feed samples. This strain completely transformed 5 mg/L ZEN within 15 h. Subsequently, a ZEN phosphotransferase gene, ZPH13, was identified in its genome. The gene was heterologously expressed in Escherichia coli BL21 (DE3), and the recombinant ZPH13 protein was biochemically characterised. The enzyme exhibited optimal activity at 35 °C and pH 8.0, achieving transformation of 2 μg/mL ZEN within 0.5 h under these conditions. Pythia was used to modify protein stability, identifying potential stabilization hotspots through free energy calculations (ΔΔG) and screening to obtain beneficial mutants N371I and N371T, which improved both enzyme activity and stability. Enzymatic transformation product analysis via ultra-high-performance liquid chromatography-quadrupole exactive-mass spectrometry revealed that ZEN was transformed into zearalenone-phosphate (ZEN-P) via phosphotransferase activity. Furthermore, co-culturing T-47D Human Breast Ductal Carcinoma (T47D) cells with the transformation products demonstrated a considerably reduced toxicity compared to that of untreated ZEN. These findings establish a new paradigm for mycotoxin transformation through innovative enzyme discovery and protein engineering strategies.