Beyond model substrates: Reevaluating enzymes claimed to hydrolyze zearalenone.

Zearalenone (ZEN) is a prevalent estrogenic mycotoxin that contaminates cereal-based food and feed and poses a substantial risk to human and animal health. Enzymatic detoxification has emerged as a promising mitigation strategy, and several α/β-hydrolases have been shown to efficiently hydrolyze ZEN (EC 3.1.1.B12). Moreover, indirect screening has identified additional structurally distinct enzymes with potential ZEN-degrading activity. In the present study, we evaluated two such enzymes-a Streptomyces exfoliatus lipase (SeLIP) and a Thermobifida cellulosilytica cutinase (TcCUT)-and compared their properties with two established zearalenone hydrolases from Clonostachys rosea and Rhodococcus erythropolis (CrZHD and ReZHD). The enzymes were characterized for thermal stability, and ZEN-degrading capacity was evaluated by direct enzymatic assay combined with LC-MS/MS. Notably, only CrZHD and ReZHD catalyzed ZEN hydrolysis, resulting in complete substrate conversion. The four enzymes were assayed for general esterase activity using p-nitrophenyl acetate (pNPA). All four enzymes exhibited measurable esterase activity toward pNPA, confirming that SeLIP and TcCUT are catalytically competent despite lacking ZEN hydrolytic activity. Structural comparison and molecular docking analyses suggested that TcCUT and SeLIP bind ZEN weakly, in non-productive conformations, likely due to the absence of a cap domain, explaining their lack of ZEN hydrolytic activity. Our results demonstrate that indirect assays based on general esterase activity cannot predict zearalenone hydrolytic activity, leading to false-positive candidate identification. Collectively, these findings highlight the value of a direct LC-MS/MS workflow for distinguishing true ZEN hydrolases from unrelated esterases, enabling more reliable candidate selection for biotechnological applications.