Study On The Detoxification Effect Of Oxygen Methyltransferase On Zearalenone

Zearalenone(ZEN)is a common mycotoxin that can have serious effects on human and animal health.Its main feature is that it has an estrogen-like effect and is extremely damaging to the reproductive system of female animals.In addition,it can cause immunosuppression,liver and kidney damage,and has potential carcinogenicity.Traditional physical and chemical detoxification methods have certain limitations.The screening of enzymes capable of degrading ZEN from microorganisms has become a hot research topic.However,the expression of ZEN degrading enzyme gene in eukaryotic vectors and structural identification of transformation products are currently available.There are few reports on the detoxification effect of animals.Previous studies have found that Cunninghamella bainieri ATCC 9244B can convert-OH on C-3 or C-5 to methoxy in ZEN molecular structure.Our group has isolated Lasiodiplodia theobromae NBRC 31059 and Hypomyces subiculosus ATCC 76476 from natural fungi which contained oxygen methyltransferase(Lt-OMT and Hs-OMT)in these two strains,which have the ability of ZEN degradation that the 3-OH and 5-OH of ZEN are converted to 3-OCH3 and 5-OCH3,respectively.This experiment evaluated the ability of oxygen methyltransferase to transform ZEN from the heterologous expression of oxymethyltransferase gene yeast,the transformation of yeast fermentation broth to ZEN,ZEN transformation product,and the identification of transformation product structure.The test evaluated the toxic effect of ZEN conversion products on mice,and provided a theoretical basis for the development of mycotoxin detoxification enzyme preparations in feed.1.Construction of an oxygen methyltransferase eukaryotic expression vector,yeast heterologous expression transformation ZENTo construct an artificial recombinant plasmid containing an oxygen methyltransferase,the oxygen methyltransferase(Lt-OMT and Hs-OMT)genes of the above two strains were obtained from NCBI,and the plasmid pRS425M(+)was used as an expression vector to construct a recombinant vector.Plasmid,transform competent Escherichia coli DH5α;screened by ampicillin(Amp)resistance,pick a single colony and incubate in a constant temperature incubator at 37 ℃ for 12-18 h,and perform restriction enzyme digestion(Nde I and Pme I)Identification,correctly inserted plasmids were named pRS425-307_YEpLeu HsOMT(307),pRS425-92 YEpLeu_LtOMT(92),pRS425-Lt-Hs-OMT_PaeciM(92-307).The correctly inserted plasmid was introduced into competent Pichia pastoris for heterologous expression.10 mg of ZEN was added to the yeast fermentation broth and transformed at 30 ℃ with a constant temperature shaker.After 48 h,the fermentation broth was extracted with ethyl acetate,evaporated on a rotary evaporator,concentrated by freezing,and the target product was detected by HPLC.The results showed that the size of the amplified fragment of Lt-OMT gene was 1200 bp,and the size of the amplified fragment of Hs-OMT gene was 1200 bp.The plasmids of No.92,No.92-307 and No.307 were enzymatically cut,and the size of the plasmids of No.92 and No.307 were 6000 bp and 1200 bp,and the size of plasmids of No.92-307 was 7579 bp,1200 bp and 771.Bp,consistent with the theoretical fragment size,demonstrates successful plasmid construction.A new product peak appeared in the ZEN yeast fermentation product by HPLC,demonstrating that the three enzymes can convert ZEN.2.ZEN conversion product purification,structure identificationIn order to study the mechanism of conversion of ZEN by oxygen methyltransferase,the crude product of ZEN transformation was purified by silica gel column,the fractions were collected,the target compound was determined by thin layer chromatography,the molecular weight of the target product was identified by LC-MS and the product structure was identified by NMR.Test Results:The conversion product was analyzed by HPLC and a new product peak appeared in the HPLC product of the conversion product compared to the ZEN standard.The retention times of ZEN standards and transformed products were 18.443 min,16.808 min,20.123 min,and 19.353 min,respectively.MS scan analysis of ZEN and transformation products with molecular weights of 317.8(M-H+),332.8(M-H+),347.8(M-H+)and 332.8(M-H+),respectively,compared to the molecular weight of the ZEN standard Adding 15,30,and 15,respectively,it is speculated that the added component may be methyl(-CH3).After NMR 1H spectrum and 13C spectrum and HMBC spectrum,the analytical data were analyzed to determine the new compounds after conversion,3-OCH3-ZEN,3,5-OCH3-ZEN and 5-OCH3-ZEN,demonstrating the mechanism of action of oxymethyltransferase is the oxymethyl modification of-OH in ZEN.3.Evaluation of detoxification effect of ZEN transformation productsTo study the detoxification effect of oxygen methyltransferase-transformed ZEN products,60 ICR mice were randomly divided into 6 groups:blank control group(physiological saline),solvent control group(10%ethanol),and ZEN treatment group(40 mg/kg),3-OCH3-ZEN group(40 mg/kg),3,5-OCH3-ZEN group(40 mg/kg)and 5-OCH3-ZEN group(40 mg/kg)by Intraperitoneal injection,once a day for 5 days,the second day after the last treatment,blood was collected from the eyeball and the animals were removed from the neck.The liver,kidney,spleen,uterus,testis and ovary were separated,and the organ coefficient was measured.The sections were used for pathological observation.The isolated blood was used for blood routine and blood biochemical assay.The cytokines IL-1β,IL-6,TNF-α,IFN-γ,IL-2,TNF-β mRNA gene expression of testis,ovary and spleen were detected by RT-PCR.Bcl-2,Bax,P-P65 and P-IκBαin testis and uterus protein expression levels were analysed by Western blot.The results showed that compared with the control group,ZEN significantly decreased the body weight,ovarian and spleen organ index(P<0.01),increased liver,kidney,uterus,testicular organ index(P<0.01).The number of white blood cells,lymphocytes and neutrophils were significantly increased(P<0.01),red blood cell count and hemoglobin content were significantly decreased(P<0.01).AST,BUN and CREA of Serum contents were significantly increased(P<0.01).TP,ALB and GLO content decreased significantly(P<0.01).ZEN group mice hepatic sinus congestion.Glomerular shrinkling,fine seminiferous tubule damage,uterine muscle thinning,gland atrophy,more blocked follicles.The levels of cytokines IL-1β,1L-6 and TNF-α were significantly increased(P<0.01).The expression levels of IL-2,IFN-γ and TNF-β were significantly decreased(P<0.01).Western-blot test showed that the expression of anti-apoptotic protein Bcl-2 in testis and uterus was significantly decreased(P<0.01),and the expression of pro-apoptotic protein Bax was significantly increased(P<0.01).The NF-κB signaling pathway protein expression of P-P65 and P-IκBα were increased significantly(P<0.01).At the same dose,3-OCH3-ZEN group,3,5-OCH3-ZEN group and 5-OCH3-ZEN showed no obvious lesions which were observed in the mice of the group.The above results indicate that ZEN(40 mg/kg)can cause strong toxic effects or even death in mice,while the same dose of the conversion products 3-OCH3-ZEN,3,5-OCH3-ZEN,5-OCH3-ZEN has no obvious side effects on mice,so it is speculated that the toxicity of ZEN after transformation is significantly reduced,and the detoxification effect is achieved.Whether the toxicity is still present at high doses,further research is needed.