Identification and characterization of thermostable hydrolases for efficient patulin degradation

Commonly present in rotten fruits, patulin (PAT) is a mycotoxin that can seriously harm human health and cause financial losses. Enzymes provide an eco-friendly, efficient, and specific approach for mycotoxin degradation. However, existing enzymes for PAT detoxification face challenges such as limited variety and poor thermostability. In this work, three PAT-degrading enzymes derived from Saccharomonospora viridis (SvDLH), Pyrinomonas methylaliphatogenes (PmDLH), and Acidimicrobium ferrooxidans (AfDLH) were identified through a sequence- and structure-based virtual screening method. All identified enzymes, which exhibit excellent thermostability (Tm > 80 °C), are capable of degrading PAT into the less toxic metabolite 2-(2-hydroxy-4-oxodihydro-2H-pyran-3(4 H)-ylidene) acetic acid, with SvDLH displaying the highest catalytic activity among them. PmDLH and AfDLH were characterized as acid-tolerant, both with optimal pH at 6. Site-directed mutagenesis confirmed that the conserved catalytic residue (Cys) is crucial for all three hydrolases to degrade PAT. In pear juice, all three enzymes (500 μg/mL) achieved 100% degradation of PAT (5 μg/mL) within 30-45 h in the absence of cofactors, with no compromise in juice quality. These enzymes are valuable candidates for the efficient detoxification of PAT and lay a technical foundation for mycotoxin removal in both food and environmental applications.