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

Yu Xia,Zhouping Wang,Yangyu Qiu,Rui He,Xiaoyuan Ma,Yahui Gao,Zifeng Wu,Qianqian Cheng

doi:10.1186/s40643-021-00395-1

Yu Xia, Zhouping Wang + Show 6 more

Open Access

https://doi.org/10.1186/s40643-021-00395-1

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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.

Highlights

Among the mycotoxins that have been widely researched, aflatoxins (AFs) are the most toxic and pose the greatest threat to human health
Construction, expression and degradation of Aflatoxin B1 (AFB1) and ZEN by ZPF1 To obtain recombinant fusion enzyme that can degrade AFB1 and ZEN simultaneously, four fusion plasmids were constructed in this work according to the methods described above
From the results shown above, it can be found that the optimized conditions for induction expression of ZPF1 were: inducing at 25 °C for 72 h, in YEPG medium containing 40.0 g/L galactose, 0.5 mmol/L MnSO4 and 0.2 mmol/L hemin

Summary

Introduction

Among the mycotoxins that have been widely researched, aflatoxins (AFs) are the most toxic and pose the greatest threat to human health. Among all the AFs analogs, aflatoxin B1 (AFB1) possesses the strongest toxicity and usually causes severe harm to human beings and animals. Zearalenone (ZEN) is another kind of mycotoxins mainly caused by Fusarium, which is commonly found in corn and rice in high temperature or warm climate regions (Lee and Ryu 2017). Most of the contaminated foods contains more than one kind of mycotoxins, and the interaction of multiple mycotoxins may cause more lethal damage (Lee and Ryu 2017; Alassane-Kpembi et al 2017). With the presence of ZEN, AFB1 has a synergistic effect on cytotoxicity in a concentrationdependent manner.

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