Food-Grade Expression of Two Laccases in Pichia pastoris and Study on Their Enzymatic Degradation Characteristics for Mycotoxins

Mycotoxin contamination poses substantial health risks to humans and animals. In this study, the two laccases PpLac1 and AoLac2 from Pleurotus pulmonarius and Aspergillus oryzae were selected and heterologously expressed in Pichia pastoris in a food-grade manner to detoxify aflatoxin B₁ (AFB₁), zearalenone (ZEN), and deoxynivalenol (DON). Both laccases exhibited degradation activity toward these three mycotoxins, while the efficiency of these for DON was relatively low. Therefore, molecular docking between these laccases and DON was conducted to analyze their potential interaction mechanisms. Furthermore, the degradation conditions of AFB₁ and ZEN by the two laccases were optimized, and the optimal degradation rates for AFB₁ and ZEN by PpLac1 reached 78.51 and 78.90%, while those for AFB₁ and ZEN by AoLac2 reached 72.27 and 80.60%, respectively. The laccases PpLac1 and AoLac2 successfully transformed AFB₁ and ZEN into the compounds AFQ₁ and 15-OH-ZEN, which were 90 and 98% less toxic than the original compounds, respectively. Moreover, the culture supernatants demonstrated effective mycotoxin degradation results for AFB₁ and ZEN in contaminated feed samples. The residual levels of AFB₁ and ZEN in all samples ranged from 6.61 to 8.72 μg/kg and 3.44 to 98.15 μg/kg, respectively, and these levels were below the limit set by the European Union standards. All of the results in this study indicated that the two laccases have excellent application potential in the feed industry.