A new Patulin-degrading enzyme from Wickerhamomyces anomalus XL1: Identification, mechanism, and application.

Patulin (PAT), a toxic mycotoxin from Penicillium species, contaminates fruits and fruit-based products, posing serious ecological and health risks. While enzymatic degradation offers an eco-friendly remediation approach, efficient degrading enzymes remain scarce. This study identified and characterized GCV2, a key PAT-degrading enzyme from Wickerhamomyces anomalus XL1, using transcriptomics, molecular docking, and molecular dynamics simulations. Transcriptomic analysis revealed oxidative stress response, detoxification, and glutathione metabolism as core PAT degradation pathways. Among 14 candidates, GCV2 showed the strongest PAT affinity (-6.31 kcal/mol). Molecular dynamics simulations confirmed GCV2-PAT complex stability, with electrostatic interactions driving binding and residues GLY607 and HIS738 providing stabilization. GCV2 was heterologously expressed and purified. Optimal conditions were 4.0 mM nicotinamide adenine dinucleotide phosphate (NADPH), pH 7.0-8.0, 30 °C, and 200 μg/mL enzyme, achieving complete degradation of 1-10 μg/mL PAT. Ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF/MS) confirmed NADPH-dependent reduction of PAT to low-toxicity E-ascladiol and Z-ascladiol without toxic by-products. Notably, GCV2 efficiently degraded PAT in pear juice, achieving 85.72% degradation within 96 h. Physicochemical analysis, electronic nose, and gas chromatography-mass spectrometry (GC-MS) revealed that GCV2 preserved the inherent quality and flavor profile of the juice while significantly increasing total phenolics and vitamin C contents. Moreover, it enhanced key aroma components, including 4-Methyl-1-pentanol and nonanal, thereby enriching the fresh fruity and citrus-like aroma characteristics. This study identified GCV2 as an efficient PAT-degrading enzyme that preserves food quality, elevates nutrition and flavor, boasting great application potential in PAT detoxification of fruit juices and other fruit-based food products.