Mycotoxin contamination of agricultural products is an essential issue regarding food safety and public health worldwide. In recent years, various methods have been employed to mitigate mycotoxins. However, they have not successfully absorbed toxins such as deoxynivalenol (DON) and related trichothecenes. One promising approach is using enzymes that can detoxify mycotoxins by altering their chemical structures. Therefore, the overall goal of this study was to produce a recombinant enzyme mixture for detoxifying DON. In the first bioinformatics phase, molecular docking between DEP A/DEP B and DON was performed to evaluate binding energies and orientations using static and dynamic simulations. For the experimental phase, the DEP A and DEP B sequences were obtained from NCBI and cloned into the pPIC9K vector for expression in Pichia pastoris GS115 yeast via electroporation. Colony PCR and electrophoresis confirmed plasmid integration into the host genome. Enzymes were expressed using BMGY and BMMY media and analyzed by SDS-PAGE and Bradford assays. An MTT assay with C2C12 cells assessed enzyme efficacy against DON toxicity. Finally, the effects of the enzymes and DON (4 mg/kg body weight) on performance, blood parameters, and intestinal histopathology were evaluated in 10-weekold male Balb/C mice. Examination of predicted protein structures indicated successful modeling. Molecular docking showed DEPs bound DON appropriately, corroborated by molecular dynamics simulations. Laboratory results demonstrated correct plasmid construction and transformation. SDS-PAGE MTT assay exhibited increased cell viability with higher enzyme levels (P<0.05). Mice receiving DON and enzymes showed improved weight gain and feed intake versus DON alone (P<0.05). Blood parameters, including leukocytes, erythrocytes, hematocrit, hemoglobin, platelets, and lymphocytes, were also improved with enzyme treatment (P<0.05). Finally, DON caused intestinal hyperemia, while the enzyme mixture prevented histopathological changes. In conclusion, these recombinant enzymes represent a promising strategy for mitigating the toxicity of mycotoxins like DON.

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