Structural and functional characterization of ZenX, a thermostable hydrolase involved in zearalenone detoxification

Zearalenone (ZEN) is an estrogenic mycotoxin, posing a serious risk to food and feed safety. In this study, a ZEN-degrading bacterium was isolated from soil and, based on phylogenetic and genomic analyses, was identified as a potential novel Microbacterium sp. and designated RD1. LC-TOF-MS/MS analysis identified a non-estrogenic hydrolysis product, indicating that RD1 degrades ZEN through lactone ring cleavage. A new hydrolase, ZenX, was cloned and heterologously expressed. It also exhibited the highest reported catalytic efficiency toward ZEN, with a specific activity of 28.06 U/mg and optimal reaction conditions of pH 9.0 and 50°C. ZenX showed high thermostability (T₅₀ = 51.9°C), which may be associated with the presence of additional intra-domain salt bridges and terminal hydrogen-bond networks. Molecular docking and sequence alignment suggested that the catalytic triad is likely composed of S112-D137-H287. Moreover, ZenX degraded ZEN in dried distillers grains with solubles, reducing its concentration from 1.1 µg/g to 0.2 µg/g. This is the first report of a ZEN hydrolase and its degradation mechanism in Microbacterium sp. Overall, these findings provide new insights into microbial ZEN degradation and the structure-function relationship of ZEN hydrolases.