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

Abstract

Aflatoxin B₁ (AFB₁) 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 AFB₁ and ZEN can be degraded by ZPF1 in reaction system 1 (70.0 mmol/L malonic buffer with 1.0 mmol/L MnSO₄, 0.1 mmol/L H₂O₂, 5.0 µg/mL AFB₁ 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 AFB₁ and ZEN, respectively), AFB₁ 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 AFB₁ 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 AFB₁ 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.

Keywords: Kluyveromyces lactis; Aflatoxin B1; Degradation; Food-grade; Fusion enzyme; Mycotoxins; Zearalenone.

Fig. 1

Flowchart for construction of fusion genes and recombinant plasmids

Fig. 2

Construction and expression of fusion enzyme ZPF1, and the degradation efficiency of AFB₁ and ZEN by ZFP1. a The electrophoresis of the PCR results of the 3 positive transformants: M: DNA marker; L1 to L3: PCR results of 3 positive GG799(pKLAC1-ZPF1) transformants. b SDS-PAGE results of culture supernatants of the 3 positive transformants: M: Protein marker; L1: K. lactis GG799 supernatant proteins; L2: K. lactis GG799(pKLAC1) supernatant proteins; L3 to L5: Three K. lactis GG799(pKLAC1-ZPF1) transformants’ supernatant protein. c Degradation results of AFB₁ (reaction system 1) and ZEN (reaction system 2) by the 3 transformants. Different letters above the histogram are significant differences (p < 0.05)

Fig. 3

Enzyme activity of PhcMnp and ZHD101.1 expressed by ZPF1 under different induction conditions. The left axes were the PhcMnp enzyme activities, and the right axes were the degradation ratios of ZEN (by ZHD101.1). a–e were the effects of time, temperature, galactose concentrations, MnSO₄ concentrations and hemin concentrations on the enzyme activities of the ZPF1

Fig. 4

Degradation of AFB₁ and ZEN by ZPF1 under different conditions in reaction system 1. a–f were the effects of time, temperature, protein concentrations, pH of buffer, concentrations of MnSO₄ and concentrations of H₂O₂. Different letters above the histogram were significant differences (p < 0.05)

Fig. 5

Degradation of ZEN by ZPF1 under different conditions in reaction system 2. a–d were the effects of time, temperature, protein concentrations and pH of buffer. Different letters above the histogram were significant differences (p < 0.05)