Enzymatic Degradation Of 3-Acetyldeoxybivalenol By Devosia SP. DDS-1 And Constructing Of The Genomic Fosmid Library

Deoxynivalenol (DON) also known as vomitoxin is one of fungi toxins by Fusarium spp. It was widespread in barley, wheat, corn, sorghum, oats and other cereal crops. Deoxynivalenol has toxicity, not only affecting food yield and quality, but also posing a risk to human and animal health, because deoxynivalenol was accumulated in human and animal body by food chain. Some physical, chemical and biological means can destroy or reduce its toxicity. Compared to traditional physical and chemical methods, biological methods have become the focus and hotspot in detoxifying mycotoxins as biological methods can overcome the drawbacks of other methods. Isolating mycotoxins-degrading bacteria by biological method and take it into practice is very useful to ensure food safety and reduce the economic losses of grain, livestock.An efficient degrading strain has been isolated in the previous studies, namely Devosia sp. DDS-1. Previous studies have indicated that DON was converted into 3-AC-DON by DDS-1, and 3-AC-DON was oxidized into 3-keto-DON, then further degradation.In this study, the enzymatic characteristics of 3-acetyl deoxynivalenol oxidase (3-AC-DON oxidase) were investigated to understand the degradation characteristics of Devosia sp. DDS-1 on 3-AC-DON, and realize the application of the enzyme. And the genomic fosmid library of Devosia sp. DDS-1 strain was constructed to provide technical platform to clone the functional genes of degradation.In this study, the enzymatic results showed that the optimal 3-AC-DON oxidase production conditions for DDS-1 were 25 ℃ pH 7.0 and 36 hours in LG medium; The optimum reaction conditions for the 3-AC-DON oxidase produced by DDS-1 were as follows:35℃, pH 7.0. The enzyme activity can be maintained above 80%from 20℃ to 40 ℃ and in the range of pH 5.0 to 9.0. Most metal ions at a concentration from 0.2mmol·L-1 to 2mmol·L-1 can promote the enzyme activity; EDTA inhibit the enzyme activity on the contrary. The results showed that the oxidase needed metal ions as auxiliary factors of enzymatic reaction; The enzyme distribution experiment showed that the 3-AC-DON oxidase in DDS-1 was endoenzyme. Crude enzyme produced by DDS-1 could degrade DON,3-AC-DON and 15-AC-DON in wheat.The genomic fosmid library of Devosia sp. DDS-1 strain was constructed in this paper. In order to obtain high quality genomic DNA of DDS-1, thee methods were used to extract genomic DNA, CTAB/NaCl method, kit method and improved kit method, respectively. The results showed that Compared to the traditional CTAB/NaCl method, the extraction method using improved kit, i.e. on the basis of the common extraction kit, together with phenol/chloroform/isoamyl alcohol, chloroform/isoamyl alcohol processes, greatly improved the quality of genomic DNA.The obtained library contained approximate 2000 clones in which the average size of insertion DNA was about 35 kb.The total capacity of this library was about 70 Mb, which covered 14 times of the genome of Devosia sp. DDS-1 (estimation of 5 Mb). Clones did not change significantly in successive transfer culture. The library could satisfy the requirements of genome sequencing and gene cloning.