Aflatoxin B₁ (AFB₁) contamination seriously threatens nutritional safety and common health. Bacterial CotA-laccases have great potential to degrade AFB₁ without redox mediators. However, CotA-laccases are limited because of the low catalytic activity as the spore-bound nature. The AFB₁ degradation ability of CotA-laccase from Bacillus licheniformis ANSB821 has been reported by a previous study in our laboratory. In this study, a Q441A mutant was constructed to enhance the activity of CotA-laccase to degrade AFB₁. After the site-directed mutation, the mutant Q441A showed a 1.73-fold higher catalytic efficiency (k_cat/K_m) towards AFB₁ than the wild-type CotA-laccase did. The degradation rate of AFB₁ by Q441A mutant was higher than that by wild-type CotA-laccase in the pH range from 5.0 to 9.0. In addition, the thermostability was improved after mutation. Based on the structure analysis of CotA-laccase, the higher catalytic efficiency of Q441A for AFB₁ may be due to the smaller steric hindrance of Ala441 than Gln441. This is the first research to enhance the degradation efficiency of AFB₁ by CotA-laccase with site-directed mutagenesis. In summary, the mutant Q441A will be a suitable candidate for highly effective detoxification of AFB₁ in the future.

Aflatoxin B₁ (AFB₁) contamination is a serious threat to nutritional safety and public health. The CotA-laccase from Bacillus licheniformis ANSB821 previously reported by our laboratory showed great potential to degrade AFB₁ without redox mediators. However, the use of this CotA-laccase to remove AFB₁ in animal feed is limited because of its low catalytic efficiency and low expression level. In order to make better use of this excellent enzyme to effectively degrade AFB₁, twelve mutants of CotA-laccase were constructed by site-directed mutagenesis. Among these mutants, E186A and E186R showed the best degradation ability of AFB₁, with degradation ratios of 82.2% and 91.8% within 12 h, which were 1.6- and 1.8-times higher than those of the wild-type CotA-laccase, respectively. The catalytic efficiencies (k_cat/K_m) of E186A and E186R were found to be 1.8- and 3.2-times higher, respectively, than those of the wild-type CotA-laccase. Then the expression vectors pPICZαA-N-E186A and pPICZαA-N-E186R with an optimized signal peptide were constructed and transformed into Pichia pastoris GS115. The optimized signal peptide improved the secretory expressions of E186A and E186R in P. pastoris GS115. Collectively, the current study provided ideal candidate CotA-laccase mutants for AFB₁ detoxification in food and animal feed and a feasible protocol, which was desperately needed for the industrial production of CotA-laccases.