Expression Of Zearalenone Degrading Enzyme And Deoxynivalenol Degrading Enzyme In Aspergillus Niger

Zearalenone(ZEN) and Deoxynivalenol(DON)are mycotoxin produced by fusarium,and the global pollution caused by them is extremely common,what’s more,they not only make huge economic losses to the world food industry but also troubled the food security of the world. Awareness and use of traditional methods to clear toxins include physical and chemical methods.Although mycotoxin can be removed by physical methods, they will not be cleared completely, and that will also make some of the original nutrients structure of the food changed, having certain drawbacks. Chemical method requires the use of certain chemical agents to achieve the purpose of removing toxins, but there are still some dangerous factors of chemical agents which haven’t been known by researchers, so there are also security issues.However, Since the biological detoxification methods have high specificity, high efficiency and has the advantage of not polluting the environment, gradually attracted so much attention。We have screened and built some ZEN and DON-degrading bacteria so far.However, there is a gap from putting into actual application due to some reasons such as low degradation ability and can not guarantee the security and so on.In this study, degrading enzyme and DON degrading enzyme was expressed by the food-grade Aspergillus niger expression system, and we tried some strategies of different signal peptides, fusion proteins and codon optimization to improve the amount of expression of ZEN and DON,hoping to obtain a safe and efficient ZEN and DON degradation bacteria.The main results of this experiment are as follows:1. Construction of expression vector p10-6-ZEN,p10-6-OZEN,p10-6-2-α-factor-ZEN,p10-6-2-α-factor-OZEN,p10-6-DON,p10-6-ODON,p10-6-2-α-factor-DON,p10-6-2-α-factor-ODON.2. By freeze-thaw method constructed expression vector was transferred into Agrobacterium, and then use Agrobacterium-mediated transformation of A. niger, obtained by PCR identified eight homologous recombinantsi, nclude p10-6-ZEN, p10-6-OZEN, p10-6-2-α-factor-ZEN,p10-6-2-α-factor-OZEN,p10-6-DON,P10-6-ODON,p10-6-2-α-factor-DON,p10-6-2-α-factor-ODONwer e identified by PCR.Among them, p10-6-ZEN, p10-6-DON were homozygous transformants and the other six were non-homozygous strains.3. 8 homologous recombinant strains obtained shake flask fermentation, the supernatant wascollected ZEN degrading enzyme gene expression strain 5-7d collected degrading enzyme gene expression DON strain 5d fermentation supernatant were detected and analyzed. The expression of its protein levels was detected by SDS-PAGE, finding that there was no obvious target ban. Western Blot test results showed that ZEN degrading enzyme gene was slight expressed.Enzyme activity test results showed that the recombination strain fermentation supernatant had a low ability of degrading for ZEN and DON.