Rational Design of a Dual Mutant Degrading Enzyme with Improved Catalytic Efficiency and Acid Tolerance for Four Aflatoxins.

Aflatoxins are major contaminants in raw food materials and their products. Developing enzymes that efficiently degrade multiple aflatoxins under acidic conditions has great practical value. In this study, the aflatoxin-degrading enzyme ADPPIII from Aspergillus terreus was systematically engineered. Substrate tunnel optimization obtained mutant F375L, which showed about twice the degradation activity for four aflatoxins versus the wild-type. Surface charge neutralization generated dual mutant F375L-K570Q with significantly improved acid tolerance. At pH 5.0, its degradation rates of aflatoxins (AFB1, AFB2, AFG1, and AFG2) were all above 76%, and it kept high degradation activity even in corn steep liquor matrix. Further analysis indicated that the F375L-K570Q mutant has enhanced substrate affinity, catalytic efficiency, structural stability, and more accurate substrate positioning. These results demonstrate that the complementary engineering of substrate tunnel residues and surface charges effectively improves catalytic performance and acid adaptability, providing a promising enzyme candidate for the biodegradation of mixed aflatoxin contamination.