Enzymatic Oxidation of Aflatoxin M₁ in Milk Using CotA Laccase

Aflatoxin M₁ (AFM₁) in milk poses a significant threat to human health. This study examined the capacity of Bacillus licheniformis CotA laccase to oxidize AFM₁. The optimal conditions for the CotA laccase-catalyzed AFM₁ oxidation were observed at pH 8.0 and 70 °C, achieving an AFM₁ oxidation rate of 86% in 30 min. The Km and Vmax values for CotA laccase with respect to AFM₁ were 18.91 μg mL^-1 and 9.968 μg min^-1 mg^-1, respectively. Computational analysis suggested that AFM₁ interacted with CotA laccase via hydrogen bonding and van der Waals interactions. Moreover, the oxidation products of AFM₁ mediated by CotA laccase were identified as the C3-hydroxy derivatives of AFM₁ by HPLC-FLD and UPLC-TOF/MS. Toxicological assessment revealed that the hepatotoxicity of AFM₁ was substantially reduced following oxidation by CotA laccase. The efficacy of CotA laccase in removing AFM₁ in milk was further tested, and the result showed that the enzyme agent achieved an AFM₁ removal rate of 83.5% in skim milk and 65.1% in whole milk. These findings suggested that CotA laccase was a novel AFM₁ oxidase capable of eliminating AFM₁ in milk. More effort is still needed to improve the AFM₁ oxidase activity of CotA laccase in order to shorten the processing time when applying the enzyme in the milk industry.