The characterization of a new stabilized lactonase from the endophytic fungi <i>Aspergillus pseudonomiae</i> with anti‐bacterial and anti‐fungal properties

Abstract Microorganisms use diverse small‐molecule signals to coordinate behavior, supporting interspecies and interkingdom communication. These chemical dialogs shape fundamental biological processes and define interactions from cooperation to antagonism. Quorum‐quenching (QQ) enzymes, which degrade or modify quorum sensing (QS) molecules such as N‐acyl‐homoserine lactones (AHLs), may play a pivotal role in shaping these networks. While bacterial QQ lactonases are well‐characterized, fungal lactonases remain undercharacterized, including their potential to target both bacterial QS molecules and fungal secondary metabolites. Using phylogenetic and structural analysis, we identified putative lactonases from the metallo‐β‐lactamase‐like lactonase family in fungi and biochemically validated the activity of one of them, from Aspergillus pseudonomiae . Structural alignment between bacterial lactonases and the AlphaFold‐predicted structure of the fungal homolog further supports that the fungal protein is indeed a lactonase. We then applied Protein Repair One‐Stop Shop algorithm for stability engineering to design a variant with improved expression in Escherichia coli and thermal stability. Purified enzyme demonstrated broad substrate specificity, effectively degrading various bacterial AHLs, with k cat / K M values of up to 1.4 × 10 5 s −1 M −1 . Both docking analysis and in vitro assays indicated that the enzyme could accommodate and act on the fungal secondary metabolite patulin, a mycotoxin associated with post‐harvest disease, showing high catalytic efficiency (1.1 × 10 4 s −1 M −1 ). When added exogenously to microbial cultures, the enzyme reduced bacterial biofilms and impaired fungal hyphal development, with the hyphal inhibition accompanied by a concentration‐dependent increase in ROS. Together, these results show that this fungal enzyme functions as a dual‐acting lactonase, positioning it as an interference enzyme in chemical signaling of fungi and bacteria and as a means to suppress pathogens and reduce patulin contamination in food.