Engineering of Bacillus Laccase frL103 for Highly Efficient Degradation of Aflatoxin B1

Aflatoxin B1 (AFB1) is a highly toxic mycotoxin that poses significant risks to food safety and public health. Bacillus laccases have shown potential for degrading AFB1, but their catalytic efficiency remains suboptimal. This study explores the structure-function relationship of Bacillus vallismortis laccase frL103 by employing molecular modeling, site-directed mutagenesis, and molecular dynamics (MD) simulations. Eight key amino acid residues in the substrate binding pocket of frL103 were selected for alanine mutagenesis. After the enzymatic properties were evaluated, site 418, a loop region near the T1 copper, was subjected to saturation mutagenesis. Site saturation mutation at T418 identified the T418A and T418S as exhibiting the highest AFB1 degradation rate, with values of 56.7 and 53.6%, 1.24 and 1.17 times higher than WT (45.7%), respectively. MD simulations showed that these mutations did not significantly affect the overall structural stability of the enzyme but changed the flexibility and hydrogen bond interactions of the loop in which they were located, thereby helping to improve substrate binding and degradation. This study provides valuable insights into the structural mechanisms underlying the enhanced degradation of AFB1 and offers a basis for designing more efficient laccase mutants for bioremediation applications.