Study On Critical Enzymes For Zearalenone Biodegradation From Acinetobacter Sp.SM04

Mycotoxin poses an imminent threat to worldwide food chain and consequently tohuman health. Zearalenone (ZEA) is a nonsteroidal oestrogenic mycotoxin mainly producedby a variety of Fusarium fungi and associated with hyperestrogenism and other severetoxicological problems of livestock and humans, leading to numerous economic losses andgreat health risks all over the world every year. Increasing attention has been paid to thedevelopment of effective and efficient strategies of ZEA detoxification. So far, variousphysico-chemical methods including adsorption, extrusion cooking and chemical treatmenthave been reported. However, these strategies may reduce nutrition components thus makingtheir commercial use limit. Now increasing interest has been generated by the possibility ofapplying biological degradation to reduce the biotoxicity of ZEA.Based on the previous findings that ZEA can be degraded into little estrogenicmetabolites by extracellular extracts of Acinetobacter sp. SM04liquid cultures, our researchfocused on the isolation of enzymes involved in ZEA degradation without H2O2fromAcinetobacter sp. SM04extracellular extracts of liquid cultures as well as the expression inrecombinant Escherichia coli.In our research, through the Sephadex G-50column isolation from Acinetobacter sp.SM04extracellular extracts of liquid cultures, Peak2fractions capable of degrading ZEAwithout adding H2O2has been isolated and the degradation rate could reach about70%.Besides, it was found that Peak2fractions had a strong alkaline tolerance and a wide sphereof pH influence on ZEA degradation as well as remaining a good performance in ZEAdecontamination at60℃.By means of SDS-PAGE and MALDI-TOF/TOF/MS analysis, four distinctive bands ofLane1(42KDa)，Lane2(30KDa)，Lane3(20KDa) and Lane4(16KDa) were separated inPeak2fractions. Then Lane1and Lane2matched the database of Acinetobacter genus withgreat homology and they were identified as outer membrane protein HMP and putative outermembrane protein respectively. On basis of the amino acid sequences of lane2(named as A4-Oxa) indentified bySDS-PAGE and MALDI-TOF-TOF/MS analysis, A4-Oxa gene from Acinetobacter sp. SM04was cloned and sequenced. Then A4-Oxa gene was ligated into the pET32a(+) expressionvector and transformed into Escherichia coli. BL21(DE3). Recombinant Escherichia coli.BL21(DE3)/pET32a(+)-A4-Oxa was successfully constructed, and further expressed by IPTGinduction. Recombinant A4-Oxa was able to degrade ZEA and its degradation rate couldreach32%. In addition, concentrations of IPTG and induction time had effects on expressionof recombinant A4-Oxa. The amount of recombinant enzyme could be maximized when theconcentration of IPTG was2.0mmol/L and induction time was6h or8h.Our study has not only greatly paved the way for further mechanism research ofAcinetobacter sp. SM04ZEA degradation, but also provided a novel and influencingreference to the development of research process of ZEA biodegradation.