Li_2026_Int.J.Biol.Macromol__150948

T-2 toxin, a widespread mycotoxin contaminating animal feed, poses serious health risks to swine. Hepatic hydrolysis serves as its primary detoxification pathway, yet the specific carboxylesterase isoenzymes involved remain unidentified. In this study, four major pig liver esterases-PLEA1, PLEB9, PLEF1, and PLEF2-were cloned and coexpressed with the chaperones GroEL/ES in E. coli Origami2 (DE3). All recombinant PLEs exhibited hydrolytic activity toward the standard substrate p-NPA and were able to selectively convert T-2 toxin to HT-2 toxin without generating other products. Among them, PLEF2 demonstrated the highest hydrolytic efficiency under optimal conditions, with a K(m) of 0.19 mM and a V(max) of 13.66 nmol/min/mg. Molecular docking and site-directed mutagenesis revealed that alanine substitutions at L101, F102 and L359 not only enhanced HT-2 toxin production but also led to the formation of neosolaniol (NEO), a less toxic hydrolytic metabolite. These findings indicate that L101, F102 and L359 are critical for substrate specificity and regioselectivity. Structural modeling suggested that the increased hydrolytic activity of the L101A and F102A variants resulted from newly formed entrance tunnels and strengthened substrate-pocket interactions, whereas the enlarged binding pocket in L359A facilitated NEO formation. This work advances our understanding of T-2 toxin metabolic detoxification in swine and identifies a novel target for mitigating its toxicity.