| Zearalenone(ZEN,ZEA)is an estrogenic mycotoxin which is produced by several Fusarium species and usually isolated from moldy corn.Due to its structural similarity to estradiol,it is able to bind to estrogen receptors in mammalian target cells and cause mammals' hyperestrogenism.Currentlly,various methods to detoxify zearalenone have been investigated which include physical,chemical and biological methods.However,these physical and chemical detoxified methods do not always work.Besides some remove essential nutrients from the feedstuff and reduce palatability.Among them,biological treatment is an attractive approach to detoxify zearalenone.Our laboratory workers isolated a strain named Acinetobacter sp.SM04 from soil which can degrade ZEN into nontoxic products with very low estrogenic activity.Then they isolated a kind of peroxidase,named A4-Prx,which could reach 130.39 mg/L and degradate 78.44% ZEN after 12 h in 30°C.Our research focused on the expression of the another enzyme(A4-Oxa).On the basis of the amino acid sequence of A4-Oxa,A4-Oxa gene from SM04 was cloned and sequenced.Then A4-Oxa was ligated into the pPIC9 K expression vector and transformed into Escherichia coli DH5?.Recombinant Escherichia coli DH5?/pPIC9K-Oxa was successfully constructed.We obtained secreted protein A4-Oxa from Pichia pastoris GS115.After the initial induction,the amount of extracellular protein was 1295.28 ?g/m L,and the expression of A4-Oxa was about 155.43 ?g/mL.A4-Oxa was found to be a nearly 38-kDa and the recombinant supernatant could degrade 53.41% ZEN after 12 h.The singal-factor experiment for the optimization of A4-Oxa expression was as follows: pH 9.0,30°C,the adittion of 0.1% Gly,1 mmol/L EDTA and 2% methanol could improve the expression of A4-Oxa protein and degradation rate of ZEN.In this case,the recombinant protein A4-Oxa content in supernatant was 220.50 ?g/m L with a ZEN degradation of 66.31%.ZEN,which is an oestrogenic mycotoxin,can induce the proliferation of MCF-7 cell in vitro.Results showed that when 20 ?g/mL ZEN was treated with the GS115/pPIC9K-A4-Oxa supernatant for 6 h,the increase of MCF-7 cell proliferation was 12.3% before which was 28.3%.It was obvious that the oestrogenic toxicity from the degradation of A4-Oxa in supernatant decreased significantly.Meanwhile,HPLC results showed that ZEN decreased almost 50% after 6 h reaction while TLC and HRMS showed that there were no new products in organic solvent.Therefore we hypothesized that the products were not in the organic phase.In addition,effects of temperature and pH on ZEN degradation activity by GS115/pPIC9K-Oxa supernatant were also examined.Results indicated that degradation rate of ZEN was increased from 33.21% to 56.12% when the temperature raised from 40°C to 80°C.And ZEN degradation rate raised from 19.63% to 51.87% when the pH of BMMY raised from 7.0 to 11.0.In conclusion,although the residual yeast in the supernatant had a certain adsorption on ZEN,high temperature or high pH was unable to make any degradation of ZEN;the recombinant A4-Oxa enzyme could play a catalytic role in either high temperature or high pH conditions,which in the future need more experiments to verify. |
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