Study On Zearalenone Degradation By Acinetobacter Sp. SM04

Zearalenone (ZEN) is a nonsteroidal oestrogenic mycotoxin produced by a variety of Fusarium fungi,which causes hyperestrogenism and related toxicoses of farm animals and humans. ZEN-contamination feeds engender numerous economic losses to animals'production, it is necessary to develop some strategies for detoxifying ZEN-contaminated foods and feeds.In this paper, a bacterium capable of efficient degrading ZEN was studied. Enzymes involved on ZEN degradation were purified and characterized in the bacterium, and these enzymes were successfully used in decontamination of ZEN-contamination feeds.A bacterium (designated SM04) which can rapidly grow on ZEN as sole carbon and energy source was isolated from agricultural soil by enrichment culture. On the basis of 16S rDNA sequencing analysis, strain SM04 has 99.5% identity with Acinetobacter genus.The ability of strain SM04 cultures in different mediums to degrade ZEN was studied. Nutrient Broth cultures of strain SM04 didn't show ZEN degradation ability, and M1 cultures of strain SM04 showed significant ZEN degradation ability. M1 medium contained the following per liter: 15 g of sodium acetate,3 g of NH4NO3, 1 g of KCl, 0.5 g of MgSO4,1.0 g of K₂HPO₄, 0.1 g of CaCl2, and10 ml of trace element (2 g/l FeSO₄·7H₂O, 0.4 g/l MnSO4·4H₂O, 0.4 g/l CuSO4·5H₂O, 0.4 g/l CoCl₆·6H₂O, and 0.5 g/l ZnCl₂).The ability of strain SM04 extracellular extracts of M1 culture for different times to degrade ZEN was also studied. Results found the extracellular extracts of M1 cultures in exponential anaphase showed most efficient ZEN degradation ability. The effects of low oxygen gas content, protease, SDS, and EDTA on ZEN degradation activity of M1 extracellular extracts were examined. Results indicated ZEN degradation activity of M1 extracellular extracts was significantly inhibited by low oxygen gas content, protease, SDS and EDTA. We inferred that oxygen gas and metal cations are required for these enzymes in the M1 extracellular extracts to degrade ZEN. In addition, effects of temperature on ZEN degradation activity of M1 extracellular extracts were also examined. Results found remained activity of ZEN degradation is nearly 60% when M1 extracellular extracts were treated with 80?for 1 h.ZEN which is an oestrogenic mycotoxin can induce the proliferation of MCF-7 cell in vitro. Result of MTT (tetrazolium salt) cell proliferation assay in MCF-7 cell line showed ZEN was degraded into little estrogenic products by M1 extracellular extracts. Few H₂O₂ were detected in the degradation products of ZEN by M1 extracellular extracts. In addition, degradation products of ZEN were extracted with ethyl acetate and analyzed by HPLC-PAD, and results revealed two new intermediate products (named as ZEN-1 and ZEN-2).M1 extracellular extracts of strain SM04 were concentrated 8 times by rotary vacuum evaporator at 50°C. Concentrated M1 extracellular extracts (10 ml) was isolated by a Sephadex G-50 column , and a ZEN oxidase fraction capable of efficient degrading ZEN was obtained from a Sephadex G-50 column isolation. The ability of ZEN oxidase fraction to degrade ZEN was also significantly inhibited by low oxygen gas content, protease, SDS, and EDTA. Result of MTT (tetrazolium salt) cell proliferation assay in MCF-7 cell line showed ZEN was degraded into little estrogenic products by ZEN oxidase fraction. Being different with degradation products of ZEN by M1 extracellular extracts, many H₂O₂ was detected in the degradation products of ZEN by ZEN oxidase fraction, and two new producnts (named as Ox-1 and Ox-2)were found in its ZEN degadation products extracted with ethyl acetate, but no ZEN-1 and ZEN-2 were detected by HPLC-PAD analysis. In addition, proteins from ZEN oxidase fraction were analyzed by SDS-PAGE and MALDI-TOF-TOF/MS. Results indicated possible cytochrome enzymes exist in ZEN oxidase fraction.M1 extracellular extracts of strain SM04 were concentrated 8 times by rotary vacuum evaporator at 50°C. Concentrated extracellular extracts (10 ml) were isolated by a Sephadex G-50 column and DEAE Sephadex A-50 column, and a thiol-redoxin peroxidase fraction was obtained. Strain SM04 thiol-redoxin peroxidase fraction showed maximal peroxidase activity in alkaline range, ZEN, Ox-1and Ox-2 can be oxidized into smaller estrogenic products by strain SM04 thiol-redoxin peroxidase fraction using H₂O₂. The optimum pH and temperature for ZEN oxidization by strain SM04 thiol-redoxin peroxidase were respectively 9.0 and 70?. After ZEN oxidized by strain SM04 thiol-redoxin peroxidase, its products were analyzed by HPLC-PAD, and three new products were detected. There were low absorption at 230-270 nm and high absorption at 300 nm for UV-Vis spectroscopy of the three new products, which indicated the benzene ring of ZEN may be cleaved.On basis of the sequences of strain SM04 thiol-redoxin peroxidase indentified by SDS-PAGE and MALDI-TOF-TOF/MS analysis, strain SM04 thiol-redoxin peroxidase gene from Acinetobacter sp. SM04 was cloned and sequenced. NCBI blast search programs analysis of thiol-redoxin peroxidase gene sequences showed strain SM04 peroxidase has high identity with 2-Cys peroxiredoxin, and it is different with these heme-peroxidases such as horseradish peroxidase and lignin peroxidase.The pET-31 b (+) vector and PCR products containing strain SM04 thiol-redoxin peroxidase ORF were both digested with NdeI and XhoI, and were purified by agarose gel electrophoresis. The gel purified strain SM04 thiol-redoxin peroxidase was ligated into the pET-31 b (+) vector using T4 DNA ligase and the ligation products were used to transform competent E. coli BL21 (DE3) cells. The thiol-redoxin peroxidase of strain SM04 was successively ligated into the pET-31(b) + expression vector and over-expressed in E. coli BL21 (DE3) by IPTG induction. Recombinant strain SM04 thiol-redoxin peroxidase showed efficient peroxidase activity and ZEN degradation ability.Model experiments on decontamination of ZEN-contaminated DDGS (dried distillers'grains and soluble) using strain SM04 M1 extracellular extracts and recombinant thiol-redoxin peroxidase was performed, and results found ZEN can be efficiently degraded by strain SM04 M1 extracellular extracts or recombinant thiol-redoxin peroxidase. H₂O₂ concentration, incubation times, temperature, and ratio of DDGS and enzymes solution (m/m) could significantly affect on ZEN degradation by strain SM04 M1 extracellular extracts or recombinant thiol-redoxin peroxidase.