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Aflatoxin B 1  (AFB 1 ) and zearalenone (ZEN) are two predominant mycotoxins ubiquitously found in corn, peanuts, and other grains, which pose a great threat to human health. Therefore, safe and effective methods for detoxification of these mycotoxins are urgently needed. To achieve simultaneous degradation of multiple mycotoxins, a fusion enzyme ZPF1 was constructed by linking zearalenone hydrolase and manganese peroxidase with a linker peptide GGGGS. This fusion enzyme was secretory expressed successfully in the newly constructed food-grade recombinant strain  Kluyveromyces lactis  GG799(pKLAC1-ZPF1), and was investigated with the mycotoxins degradation efficiency in two reaction systems. Results showed that both AFB 1  and ZEN can be degraded by ZPF1 in reaction system 1 (70.0 mmol/L malonic buffer with 1.0 mmol/L MnSO 4 , 0.1 mmol/L H 2 O 2 , 5.0 µg/mL AFB 1  and ZEN, respectively) with the ratios of 46.46% and 38.76%, respectively. In reaction system 2 (50.0 mmol/L Tris–HCl, with 5.0 µg/mL AFB 1  and ZEN, respectively), AFB 1  cannot be degraded while ZEN can be degraded with the ratio of 35.38%. To improve the degradation efficiency of these mycotoxins, optimization of the induction and degradation conditions were fulfilled subsequently. The degradation ratios of AFB 1  and ZEN by ZPF1 in reaction system 1 reached 64.11% ± 2.93% and 46.21% ± 3.17%, respectively. While in reaction system 2, ZEN was degraded by ZPF1 at a ratio of 41.45% ± 3.34%. The increases of degradation ratios for AFB 1  and ZEN in reaction system 1 were 17.65% and 7.45%, respectively, while that for ZEN in reaction system 2 was 6.07%, compared with the unoptimized results.

Simultaneous degradation of two mycotoxins enabled by a fusion enzyme in food-grade recombinant Kluyveromyces lactis