| Zearalenone(ZEN)),also known as F-2 mycotoxin,is a potent estrogenic metabolite produced by some Fusarium and Gibberella species,which can be found mainly from mildew of corn or other crops and feed.ZEN and its metabolites are similar to the endogenous estrogen structure in humans and animals,which indicated they can compete with estrogen receptors and trigger reproductive toxicity.The wide distribution and high harmfulness of ZEN bring great economic losses to agricultural industry and pose a serious threat to human health.In order to reduce the harm caused by ZEN,scholars worldwide have studied various methods of detoxification,such as the physical method represented by adsorbents and anti mildew agents,chemical methods based on acid and alkali treatment and ozone treatment,and biodegradation which now mainly focus on the research of ZEN degradation strains and their key enzymes.At present,the most advanced ZEN adsorbents are not efficient enough for their adsorption.Chemical treatment is harmful to the raw materials and also the environment.Therefore,biodegradation has gradually become the main research direction of the degradation of ZEN.The research group has successfully screened a strain of Acinetobacter sp.SM04 from mildew soil which can efficiently degrade ZEN.Two kinds of enzymes with degradation efficiency are isolated from the culture medium of SM04,of which the oxidase A4-Oxa has been induced in the Pichia pastoris expression system.It can produce 155.43 ?g/mL protein and remove ZEN 53.41% after 12 hours treatment at 40?.This paper mainly studies peroxidase Prx.By designing primers P1 and P2,the encoding gene of A4-Prx from SM04 was cloned and His-tag was decided to be linked to the 3'.The target gene was inserted into pPIC9 K by genetic engineering method and transferred to Escherichia coli.DH5?,and the pPIC9K-Prx is constructed successfully.pPIC9K-Prx was transferred into Pichia Pastoris GS115,and the recombinant Pichia pastoris GS115/pPIC9K-Prx-His was constructed and it can produce protein 167.31 ?g/mL protein.The fermentation supernatant could degrade 99.43% ZEN after 12 hours treatment under60?.In order to verify whether the inserted His-tag affects the activity of Prx enzyme,the effect of Prx-His and A4-Prx on the degradation rate and peroxidase activity of ZEN was tested by setting different pH and temperature.The results showed that the insertion of His-tag did not produce significant adverse effects on Prx protein,and its heat resistanceincreased slightly.The optimum temperature of Prx-His is about 60?,and the optimum pH is about 8?After the precipitation of ammonium sulfate and the elution of Ni-Sepharose 6 FF column,the peak 3 with high peroxidase activity was collected.The purification process was monitored.The purification multiple was 9-fold.In order to verify the structure of ZEN degradation products,the product of 6.5 minute peak under the liquid phase was prepared by semi preparation liquid chromatography.The result of high resolution mass spectrometry compared with the control group showed that the ratio of the product was 380.In addition,the MTT experiment was used to test the cell proliferation rate of MCF-7cells.The results showed that the proliferation rate of the cells after the enzyme treatment decreased significantly from 275% to 150%,and the cell proliferation rate decreased with the increase of treatment time,from 230% to 101%.It was proved that ZEN was degraded to low estrogenic toxic products.This paper also used ZEN derivative ?-ZOL and ?-ZOL as substrate to determine the degradation rate.As a result,?-ZOL was almost completely degraded,while the degradation rates of ?-ZOL and ZEN were 62.19% and 74.87%respectively after 8 hours treatment under 60?. |
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