Characterization of a Novel CotA-Type Laccase from the Safe Bacillus velezensis T-3 Via Genome Analysis, Quantitative Proteomics and Molecular Docking

Laccase is a class of known copper-dependent oxidase which can antagonize molds and degrade mycotoxins efficiently, but few reports exist on the structure and function of laccase from Bacillus velezensis. In this study, we identified a CotA-type laccase gene (locus tag: ctg_03438) in B. velezensis T-3, and its DON removal capacity was investigated via bioinformatics analysis, 4D-DIA quantitative proteomics, and in vitro fermentation assays. Results indicated that the CotA gene encodes an extracellular spore coat protein (CotA) that lacks typical signal peptides and transmembrane domains. Crucially, 4D-DIA quantitative proteomic analysis confirmed that CotA in B. velezensis T-3 is expressed at a high level, with 23 unique peptides covering 47.30% of the protein sequence, including a strictly proteotypic peptide (PLKEADTSRK). Molecular docking revealed a stable DON-CotA interaction (ΔG = -6.299 kcal/mol) with a 6.4 Å distance to the T1 copper center, suggesting a potential binding mode for DON recognition. Functionally, B. velezensis T-3 demonstrated potent antifungal activity (inhibition zone: 3.51 cm against Fusarium graminearum (F. graminearum)) and achieved a 30.06% reduction of DON in contaminated feed within 10 days. This study provides a safe candidate strain and molecular-level evidence for the development of biodetoxification agents.