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The resistance of feed enzymes against proteases is crucial in livestock farming.In this study,the trypsin resistance of aflatoxin-detoxifizyme(ADTZ)is improved.ADTZ possesses 72 lys/arg residue sites,45 of which are scattered on the outermost layers of the molecule(RSA≥ 25%).These 45 lys/arg sites could be target sites for trypsin hydrolysis.By considering shape-matching(including physical and secondary bond interactions)and the “induced fit-effect”,we hypothesized that some of these lys/arg sites are vulnerable to trypsin.A protein-protein docking simulation method was used to avoid the massive computational requirements and to address the intricacy of selecting candidate sites,as candidate site selection is affected by space displacement.Optimal mutants(K244Q/K213C/K270T and R356E/K357T/R623C)were predicted by computational design with protein folding energy analysis and molecular dynamics simulations.A trypsin digestion assay was performed,and the mutants displayed much higher stability against trypsin hydrolysis compared to the native enzyme.Moreover,temperature-and pH-activity profiles revealed that the designed mutations did not affect the catalytic activity of the enzyme.