CN109136300A - A kind of zearalenone poison-removing method - Google Patents

A kind of zearalenone poison-removing method Download PDF

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CN109136300A
CN109136300A CN201810964751.3A CN201810964751A CN109136300A CN 109136300 A CN109136300 A CN 109136300A CN 201810964751 A CN201810964751 A CN 201810964751A CN 109136300 A CN109136300 A CN 109136300A
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omt
leu
zearalenone
zen
supernatant
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CN109136300B (en
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李金贵
刘明江
时从来
韩晴
韩一晴
徐玉泉
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Yangzhou University
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    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
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    • C12P17/08—Oxygen as only ring hetero atoms containing a hetero ring of at least seven ring members, e.g. zearalenone, macrolide aglycons
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Abstract

The invention belongs to field of biotechnology, it is related to a kind of zearalenone poison-removing method, the gene of Lt-OMT is connect to building 92-Lt-OMT-YEp-Leu of Recombinant Artificial plasmid with pRS425 carrier, artificial 92-Lt-OMT-YEp-Leu of plasmid is transferred in competence yeast cells, yeast cells is placed in Leu auxotroph culture medium and is cultivated, zearalenone ZEN is added in Leu auxotroph culture medium, zearalenone ZEN is converted 3-OCH by the Lt-OMT of yeast cells synthesis3 - ZEN.ZEN of the invention is converted into 3-OCH by Lt-OMT3 - ZEN, experiments have shown that 3-OCH3 - ZEN is to mouse without obvious toxic-side effects.Lt-OMT analyzes the transformation efficiency of ZEN up to 98% or more, HPLC and remains almost without substrate in yeast fermentation system.

Description

A kind of zearalenone poison-removing method
Technical field
The invention belongs to field of biotechnology, are related to a kind of zearalenone (Zearalenone, ZEN) poison-removing method, This method utilizes the oxygen transmethylase of a kind of Lasiodiplodia theobromae (Lasiodiplodia theobromae) (Lasiodiplodia theobromae Oxymethyltransferase, Lt-OMT) is by 3 of zearalenone ZEN Hydroxyl (- OH) is converted into oxygen methyl (- OCH3)。
Background technique
Zearalenone (ZEN) is mainly Fusarium graminearum (Fusarium graminearum), fusarium tricinctum (F.tricinctum), yellow fusarium (F.culmorum), scouring rush's fusarium (F.equiseti), F.semitectum (F.sernitectum), the mycetogenetic secondary metabolite such as fusariun solani (F.solani), be widely present in cereal and its In product.Zearalenone ZEN has estrogenic effect, can induce reproduction poison in conjunction with animal body estrogen receptor Property and teratogenesis, carcinogenesis, seriously affect animal reproduction performance, in addition, zearalenone ZEN can lead to immunity of organism suppression The harm such as system, lesions of liver and kidney, Disorder of Digestion and A orption.
China's plant of grain crops, purchase, process scale degree be not high at present, personal or small business crop or its Mould prevention awareness is not strong during product processing, transport or storage, leads to zearalenone ZEN recall rate in Related product It is higher, and average content Yuan Chao developed country average level.Such as: 2017, feedstuff or the feeding to more than China such as Gong Aqiong Mycotoxin detection and analysis obtain in material, and ZEN recall rate is 91.9% in corn, exceeding standard rate 5.4%；ZEN is detected in wheat bran Rate is 100%, exceeding standard rate 13%；Distiller's dried grain and its soluble matter (Distillers Dried Grains with Solubles, DDGS) in ZEN recall rate be 100%, exceeding standard rate 43.8；ZEN recall rate is 100% in corn germ cake, is surpassed Mark rate is 35%；ZEN recall rate is 100% in pannage, exceeding standard rate 7.1%；Ruminating ZEN recall rate in material is 100%, is surpassed Mark rate is 22.2%；By the way that ascendant trend is presented with the exceeding standard rate of former years data comparison, ZEN.Therefore, zearalenone ZEN Pollution causes resource huge waste, seriously endangers the development of China's animal husbandry and seriously threatens to the generation of people's life and health.
At this stage, ZEN poison-removing method mainly has physical method, chemical method and bioanalysis three classes.Physical method is adsorbed using toxin Agent carries out detoxification, but most adsorption effects are poor, detoxification is not thorough, higher cost, and feed nutrient loses larger, the day of one's doom Its application in feed detoxification is made.Chemical method is better than physical method, but the same nutritional ingredient because caused by ZEN detoxification efficiency It destroys or secondary pollution is caused not to be used widely.Efficient degradation of the enzyme that bioanalysis mainly utilizes microorganism to generate to ZEN Effect, or removes the specific adsorption of ZEN using microbial body, which has that reaction condition is mild, specific good, nothing Chemical agent residue does not destroy the advantages that feed nutrition, is processing most promising in current mycotoxin detoxification technology Technology.But there is also deficiencies for the technology, and the microorganism of such as screening is also animal pathogen；The secondary metabolite that microorganism generates To animal body, there are toxic effects；The intermediate product of bio-enzyme degradation ZEN is to the still toxic effect of animal body；Biological production of enzyme It is low, isolate and purify process complexity, unstable, the enzyme effect condition harshness of enzymatic activity etc., these disadvantages also limit ZEN biological detoxication The development in field.
Summary of the invention
In order to overcome drawbacks described above, the content of present invention, which provides one kind, has good detoxification effect, and detoxification is high-efficient, nontoxic pair 3-OH of zearalenone (ZEN) are converted to-OCH using Lt-OMT by the zearalenone poison-removing method of effect3。
In order to achieve the above-mentioned object of the invention, the technical solution adopted by the present invention are as follows: a kind of zearalenone poison-removing method, It is characterized by: the oxygen methyl modification enzyme Lt-OMT using Lasiodiplodia theobromae replaces 3 hydroxyl-OH of zearalenone It is changed to oxygen methyl group-OCH3。
The gene of Lt-OMT is connect to building artificial recombination plasmid 92-Lt-OMT-YEp-Leu with pRS425 carrier, by people Work recombinant plasmid 92-Lt-OMT-YEp-Leu is transferred in competence yeast cells, and yeast cells is placed in the training of Leu auxotroph It supports and is cultivated in base, zearalenone is added in Leu auxotroph culture medium and realizes co-cultivation, yeast cells synthesis Zearalenone is converted 3-OCH by Lt-OMT3- ZEN, reaction equation are as follows:
Zearalenone poison-removing method, specific steps are as follows:
(1) gene cloning:
Normal chain and negative strand primer are designed according to the nucleotide sequence of Lt-OMT, by the method for PCR, obtains target gene Lt-OMT；
(2) building of recombinant plasmid:
1. utilizing restriction enzyme NdeI, PmeI digestion Lt-OMT PCR product and pRS425 carrier, connected by T4DNA It connects enzyme and Lt-OMT genetic fragment after digestion is connect construction recombination plasmid 92-Lt-OMT-YEp- with pRS425 carrier after digestion Leu；
It is expressed 2. recombinant plasmid 92-Lt-OMT-YEp-Leu is imported in competent E.coli DH5 α cell, alkaline lysis Whether method extracts plasmid, complete using agarose electrophoresis verifying purpose gene is carried out after NdeI and PmeI digested plasmid；
(3) yeast conversion:
The 92-Lt-OMT-YEp-Leu recombinant plasmid of building is imported in competence yeast cells BJ5464-NpgA；
(4) zearalenone and yeast transformant co-culture:
10mg zearalenone is placed in the auxotroph-Leu culture solution containing yeast transformant and cultivates 48h； The yeast transformant is the 92-Lt-OMT-YEp-Leu recombinant plasmid of 1 μ g；
(5) tunning extracts:
1. fermentation liquid 1580g is centrifuged 5min, supernatant, sediment are collected respectively；
2. 1. middle supernatant is mixed with isometric ethyl acetate, is poured into separatory funnel after mixing well, stand and divide Layer collects supernatant, sediment respectively；
3. the sediment of step 2. is uniformly mixed with isometric ethyl acetate, stratification, supernatant is collected respectively, is sunk Starch；
4. the sediment of step 3. is uniformly mixed with isometric ethyl acetate, stratification, supernatant is collected respectively, is sunk Starch；
5. 2. 3. 4. supernatant mixes and be transferred to Rotary Evaporators by step, it is concentrated into paste；
6. 3 times of volumes methanols and ultrasound 15min are added in 1. sediment, pour into separatory funnel, stratification, respectively Collect supernatant, sediment；
7. by step, 6. sediment is uniformly mixed with isometric ethyl acetate, is extracted three times repeatedly, is merged supernatant three times；
8. by step, 7. supernatant is transferred to Rotary Evaporators, is concentrated into paste；
9. 5. 8. concentrate that step is obtained chromatography methanol redissolves, after 0.22 μm of membrane filtration, 13400g centrifugation 10min, supernatant are fitted into spare in brown liquid-phase inlet bottle.
(6) converted product is purified:
Each fraction is collected with each component of the mobile phase separation crude extract of different proportion by silica gel column chromatography；It will be each Fraction progress thin-layer chromatography (Thin-layer chromatography, TLC) and contact plate, screen target compound.
(7) converted product structural analysis:
Tunning is through high performance liquid chromatography (High Performance Liquid Chromatography, HPLC) Analysis finds that the ZEN substrate peak of 4.790min disappears, and generates a new peak in 4.175min (see attached drawing 3).
It collects converted product fraction (compound at 4.175min), rotation steaming method is precipitated and weighs to white crystalline powder, Show that noval chemical compound quality is 9.8mg.
Molecular weight through mass spectrometer (Mass Spectrometry, MS) analysis noval chemical compound is 333.8 (see attached drawing 4)。
It is obtained through nuclear magnetic resonance method (Nuclear-magnetic Resonance, NMR) analysis, new compound structure 3- OCH3- ZEN (see attached drawing 5, table 2).
Analysis show that the enzyme is 98% to the transformation efficiency of ZEN in yeast fermentation system (BJ5464-NpgA).
The nucleotide sequence of Lt-OMT is as shown in SEQ ID NO.1, and amino acid sequence is as shown in SEQ ID NO.2.
Compared with the existing technology, the invention has the benefit that
1.ZEN is converted into 3-OCH by Lt-OMT3- ZEN, test prove 3-OCH3- ZEN is to mouse without obvious toxic-side effects.
2. Lt-OMT analyzes almost without substrate the transformation efficiency of ZEN up to 98% or more, HPLC in yeast fermentation system Residual.
The present invention is used in yeast fermentation system (Saccharomyces cerevisiae BJ5464-NpgA, BJ5464- NpgA heterogenous expression Lt-OMT in), Lt-OMT can specificity convert-OCH for 3-OH of ZEN3, transformation efficiency is up to 98% More than.Above-mentioned converted product is subjected to mouse toxicity test after purification, as a result, it has been found that compound is connected by 10mg/kg dosage after conversion Continuous administration 5 days, to mouse without obvious toxic-side effects, and can obviously cause mouse liver, kidney, reproductive system with the ZEN of dosage Damage, the death rate illustrate that the method has good detoxification effect to ZEN, detoxification is high-efficient up to 50%.
Detailed description of the invention
Fig. 1,92-Lt-OMT-YEp-Leu plasmid map；
Target gene integrity check figure after Fig. 2,92-Lt-OMT-YEp-Leu plasmid enzyme restriction, wherein 1: target gene；2/ 3: digestion products；
The HPLC analysis result figure of Fig. 3, ZEN, converted product, note: A:ZEN standard items HPLC map；B: converted product HPLC map；
The mass spectrometry results figure of Fig. 4, ZEN, converted product, note: A:ZEN standard items MS map；B: converted product MS figure Spectrum；
The NMR of Fig. 5, ZEN converted product analyzes result figure, note: A:3-OCH3-ZEN 1H spectrogram；B:3-OCH3-ZEN 13C Spectrogram；C:3-OCH3- ZEN HMBC map；D:3-OCH3- ZEN HSQC map.
The nucleotide sequence of table 1, Lt-OMT；
The NMR spectra parsing of table 2, ZEN converted product.
Specific embodiment
The invention will be further described with attached drawing combined with specific embodiments below.
(1) experimental material:
1. bacterial strain and carrier:
E.coli DH5 α competent cell is ShiJi Co., Ltd purchased from health；
BJ5464-NpgA: Biological Technology institute, Chinese Academy of Agricultural Sciences is derived from；PRS42 carrier: by Chinese agriculture section The building of institute's biotechnology research institute Biotechnology Compose Experiment room.
2. enzyme and kit:
Nde I, Pme I, T4 ligase are purchased from NEB Products；
Taq enzyme is purchased from Quan Shi King Company；
Plasmid extracts, plastic recovery kit is purchased from TIGEN company, AXYGEN company respectively.
Other test method without specific conditions in embodiment, conventionally carry out, and molecular cloning presses (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in laboratory manual, or according to system Make condition proposed by manufacturer.
(2) specific embodiment of the .Lt-OMT to the methylation modification of ZEN:
1. target gene is cloned:
According to the nucleotide sequence (table 1, SEQ ID NO.1) of Lt-OMT, design primer obtains mesh by the method for PCR Gene.Lt-OMT primer sequence, normal chain primers F: 5 '-GATCTGGGTGGTTATGCAAGATA-3 ' (SEQ ID NO.3)；Instead Strand primer R:5 '-ACCATCTGCTGACTTGTACGA-3 ' (SEQ ID NO.4), Size:1.2kb.
1 Lt-OMT nucleotide sequence information of table (SEQ ID NO.1)
Note: the amino acid sequence (SEQ ID NO.2) of the nucleotide sequence of Lt-OMT are as follows:
MRSYSLDIVPKIEAILADAKQLEDEDLSARLGLMTQVDRLYQDLESPIELFFRQWTDVTVYSCVNVAIK LGIFEKMKAKENISAQELADLVGVDLDVIVRVMRVLVAAGIVASPVEETYSHTPKSLIFVRGKGTAVGSFELIALYA KSYLSLPEYLKTRPSADLYDVCKTPYAYEYGIEGKPFYEALSSVPQHLDIFNRAMDEPTTECGIFPWSSLKEEVQAE PDRAFIVDIGGGSGKVLHFARAETGDVFGTSARLILQERPDALAQLDTAQQSGIEMMAYDFHTEQPIKGAHIYHLGH ILHNHPDHICRDILKRIAEAMSPKSRLLIFDAVIPARTEPGKYMNGYLIDIVGLATGGKERTEKETAALLDAEGLEF VRVWHGQAGWQGVIEARLKRG
1. PCR reaction system (25 μ L) is as follows:
Template DNA (0.1 μ g/mL): 1.00 μ L；
Positive strand primer (100pmol/L): 2.00 μ L；
10 × Buffer (1.0mmol/L): 2.50 μ L；
Negative strand primer (100pmol/L): 2.00 μ L；
DNTP (1.0mmol/L): 8.00 μ L；
Taq archaeal dna polymerase (5.0U/ μ L): 1.00 μ L；
Sterilize distilled water: 8.50 μ L.
2. PCR reaction condition are as follows: 94 DEG C of initial denaturations handle 1min；94 DEG C of denaturation 30s, 50 DEG C of annealing 40s, 72 DEG C extend 90s, cycle-index 30 times；Last 72 DEG C of extensions 7min, saves backup in 4 DEG C.
3. PCR product is carried out 1.0% agarose gel electrophoresis detection, and carry out PCR product recycling.
2. construction of recombinant plasmid:
1) with Nde I and Pme I digestion Lt-OMT (PCR product that step 1 obtains) and PRS425 carrier.
2) using digestion products in the connection 1) of T4DNA ligase to construct 92-Lt-OMT-YEp-Leu plasmid (see attached drawing 1)。
3) 92-Lt-OMT-YEp-Leu plasmid is converted into E.coli DH5 α, and even spread is to containing 50 μ g/mL ammonia In solid bacteriolyze meat soup (Luria-Bertani, the LB) culture medium in benzyl XiLin (Ampicillin, Amp), it is placed in 37 DEG C of incubators Middle culture 12-14h.
4) picking positive clone molecule is transferred in the LB liquid medium (4mL) containing Amp (50 μ g/mL), and 37 DEG C of constant temperature shake Bed (200rpm) cultivates 12-14h.
5) alkaline lysis method of extracting plasmid is utilized, with Nde I and Pme I digested plasmid, generates two silver of 6000bp, 1200bp Section (attached drawing 2).
6) plasmid is saved.
3. yeast conversion:
The 92-Lt-OMT-YEp-Leu plasmid (step 2 acquisition) of building is transformed into BJ5464-NpgA, specific steps It is as follows:
1) powder peptone dextrose culture-medium (Yeast Extract Peptone Dextrose is leached in yeast solid Medium, YPD) on culture medium, BJ5464-NpgA single colonie is separated by method of scoring, is placed in (30 DEG C) of constant incubator cultures 16h。
2) picking single colonie is inoculated in YPD fluid nutrient medium, sets in constant-temperature table (30 DEG C, 220rpm) cultures, every 2h detects OD value, works as OD600Stop culture when between 0.8-1.0.
3) saccharomycete is collected in centrifugation (500g, 4min), abandons supernatant.
4) it is converted using Yeast Transformation Kit (ZYMO, No.T2001), concrete operations are as follows:
A. cell is resuspended with 10mL EZ1 solution, supernatant is abandoned in centrifugation (500g, 4min) afterwards.
B. cell is resuspended with 1mL EZ2 solution, conversion or -80 DEG C of preservations is carried out after packing (50 μ L).
C. 500 μ L are added after the 92-Lt-OMT-YEp-Leu plasmid of 1 μ g being mixed with the competent yeast cells of 50 μ L EZ3 solution is uniformly mixed, and is placed in constant incubator (30 DEG C) and is incubated for 45min, is resuspended 2-3 times during being incubated for.
5) it draws 100 μ L conversion fluids to be coated onto solid leucine shortage culture medium (- Leu), 30 DEG C of constant temperature incubation 72h.
6) bacterium colony grown is yeast transformant.
4. yeast transformant and ZEN are co-cultured:
1) yeast transformant (acquisition of step 3 yeast conversion) is applied to by new solid SC-Leu culture medium by method of scoring On (Clontech, 3013C204), 30 DEG C of constant temperature incubation 48h.
2) it collects yeast cells and is inoculated into 250mL liquid-Leu culture medium, be placed in constant-temperature table (30 DEG C, 220rpm) Middle expansion culture is for 24 hours.
3) continue to cultivate after 250mL YPD culture medium, 1mL ZEN standard solution (10mg/mL) being added in conical flask 48h。
5. tunning extracts:
1. fermentation liquid (step 4: yeast transformant and ZEN are co-cultured and obtained) 1580g is centrifuged 5min, supernatant is collected respectively Liquid, sediment.
2. 1. middle supernatant is mixed with isometric ethyl acetate, is poured into separatory funnel after mixing well, stand and divide Layer collects supernatant, sediment respectively.
3. the sediment of step 2. is uniformly mixed with isometric ethyl acetate, stratification, supernatant is collected respectively, is sunk Starch.
4. the sediment of step 3. is uniformly mixed with isometric ethyl acetate, stratification, supernatant is collected respectively, is sunk Starch.
5. by step, 2. 3. 4. supernatant mixes and is transferred to Rotary Evaporators, be concentrated into paste (37 DEG C of water-bath constant temperature, it is cold Solidifying 0 DEG C of pond constant temperature, vacuum pressure constant pressure 100mbar, rotary evaporation revolving speed are 60rpm).
6. 3 times of volumes methanols and ultrasound (frequency 39900Hz) 15min are added in 1. sediment, separatory funnel is poured into In, stratification collects supernatant, sediment respectively.
7. by step, 6. sediment is uniformly mixed with isometric ethyl acetate, extract repeatedly three times (with step 2. -4.), close And supernatant three times.
8. by step, 7. supernatant is transferred to Rotary Evaporators, is concentrated into paste (5. with step).
9. 5. 8. concentrate that step is obtained is redissolved with chromatography methanol (2mL), after 0.22 μm of membrane filtration, 13400g It is centrifuged 10min, supernatant is fitted into spare in brown liquid-phase inlet bottle.
6. purifying converted product
1. experimental material: 200 mesh silicagel columns；Thin-layer chromatography (Thin-layer chromatography, TLC) and silica gel plate； Hand-held ultraviolet lamp.
2. method: by silica gel column chromatography, separating crude extract with the mobile phase (methylene chloride, methanol) of different proportion Each component collects each fraction；Each fraction is subjected to TLC contact plate, (mobile phase is methylene chloride to screening target compound: methanol body Product is than being 99:1).
7. the HPLC of converted product is analyzed, is prepared:
1) analysis condition is as follows:
HPLC instrument (Agilent 1290 Infinity UHPLC, GER)；Chromatographic column: ZORBAX Eclipse Plus C18 Narrow Bore,5μm×2.1mm×150mm。
Mobile phase: acetonitrile: water (90:10) carries out gradient elution；
Condition of gradient elution: 0~1min, acetonitrile 10%；1~3min, acetonitrile is from 10% → 95%；3~5min, acetonitrile It is 95%；5~8min, acetonitrile is from 95% → 10%；8~10min, acetonitrile 10%.
Flow velocity: 0.5mL/min, Detection wavelength: 300nm.
2) interpretation of result:
1. the ZEN substrate peak of retention time 4.790min disappears, retention time 4.175min generates a new peak (see attached drawing 3)。
2. collecting converted product fraction (compound at 4.175min), (37 DEG C of water-bath constant temperature, condensation reservoir is permanent for rotary evaporation 0 DEG C, vacuum pressure constant pressure 100mbar of temperature, rotary evaporation revolving speed are 60rpm) it is precipitated and weighs to white crystalline powder, it obtains new Compound quality is 9.8mg.
8. the MS of converted product is analyzed:
1) analysis condition is as follows:
A) instrument and equipment:
MS instrument (Agilent 6100Single quadrupole mass spectrometry, GER)；UHPLC instrument (Agilent 1290Infinity UHPLC,GER)；Chromatographic column: ZORBAX Eclipse Plus C18 Narrow Bore, 5 μm×2.1mm×150mm。
B) electrospray ionisation (Electrospray ionization, ESI) condition:
Capillary voltage: 3.0kV；Source temperature: 20 DEG C；Remove solvent temperature: 350 DEG C；Taper hole air-flow: nitrogen, flow velocity 100L/ h；Go solvent stream: nitrogen, flow velocity 600L/h；Collision gas: argon gas collides air pressure 2.60 × 10-4Pa。
C) scanning mode:
Anion scanning.
D) multiple-reaction monitoring (Multiple reaction monitoring, MRM) condition:
Scanning range (m/z): 0-1000.
2) interpretation of result:
ZEN standard items: residence time: 0.2s；Orifice potential: 30V；Collision energy: 28eV, 32eV；Retention time: 4.790min.Parent ion (m/z): 317.8, daughter ion (m/z): 300.8 (see attached drawing 4A).
Converted product: residence time: 0.2s；Orifice potential: 30V；Collision energy: 25eV, 30eV；Retention time: 4.175min.Parent ion (m/z): 332.8, daughter ion (m/z): 314.8 (see attached drawing 4B).
The molecular weight of converted product is that 333.8, ZEN molecular weight is 318.8, increase of the molecular weight compared with ZEN of converted product 15.
9. the NMR (Bruker, AVANCE 600MHz, GER) of converted product is analyzed:
1) analysis condition is as follows:
A) instrument and equipment:
NMR instrument (Bruker, AVANCE 600MHz, GER).
B) parameter is analyzed:
The circulation waiting time is 2s, incorporation time 100ms, and 4 μ s of pulse delay time (t1), spectrum width 10000Hz are adopted The sample time is 1.64s, sampling number 32k, accumulative frequency 64.
Proton Resonance Frequency is 600.13MHz.1H-1The F of H J-RES2(1H) spectrum width tieed up is 600.13Hz, sampled data Dot matrix t2×t1=4096 × 64；1H-13The F of C HSQC2(1) and F H1(13C) tie up spectrum width difference 6313.13Hz and 26409.97Hz, sampled data dot matrix t2×t1=2048 × 90；1H-13The F of C HMBC2(1) and F H1(13C the spectrum width difference) tieed up 6313.13Hz and 33201.94Hz, sampled data dot matrix t2×t1=2048 × 100.
2) interpretation of result:
Confirm that-the OH on 3 C of ZEN is converted into-OCH after structure elucidation3(see attached drawing 5, table 2).Prove Lt-OMT pairs 3-OH of ZEN carry out the modification of oxygen methyl.
Table 2, the parsing of ZEN converted product NMR spectra
Note: s=single, m=multiple, t=triple, CD3OD are deuterated methanol, and δ is chemical shift, and J is coupling Constant is closed, ppm is chemical shift unit.
Example of the present invention is the description of the invention and cannot limit the present invention, with the comparable meaning of the present invention Any change and adjustment in range, are all considered as within the scope of the invention.
Sequence table
<110>Yangzhou University
<120>a kind of zearalenone poison-removing method
<130> xhx2018072601
<141> 2018-07-26
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1197
<212> DNA
<213> Lasiodiplodia theobromae
<400> 1
atgagatcct actcattgga tatcgtccca aagattgaag ctattttggc tgatgctaag 60
caattggaag atgaagattt gtctgctaga ttgggtttga tgacccaagt tgacagatta 120
taccaagact tggaatcccc aatcgaatta ttcttcagac aatggactga tgtcaccgtt 180
tactcttgtg ttaatgttgc cattaagttg ggtatcttcg aaaagatgaa ggccaaagaa 240
aacatctccg ctcaagaatt ggctgatttg gttggtgttg atttggatgt tatcgtcaga 300
gttatgagag ttttggttgc tgctggtata gttgcttctc cagttgaaga aacttactct 360
catactccaa agtccttgat cttcgttaga ggtaaaggta ctgctgttgg ttcctttgaa 420
ttgattgcct tgtacgctaa gtcctacttg tctttgccag aatacttgaa aactagacca 480
tctgctgact tgtacgatgt ctgtaaaact ccatacgctt acgaatacgg tattgaaggt 540
aagccattct acgaagcttt atcctctgtt ccacaacact tggatatttt caacagagct 600
atggatgaac ctactaccga atgtggtatt tttccatggt cctcattgaa agaagaagtt 660
caagctgaac cagatagagc cttcatagtt gatattggtg gtggttctgg taaggtcttg 720
cattttgcta gagctgaaac tggtgatgtt tttggtactt cagctagatt gatattgcaa 780
gaaagaccag atgctttggc tcaattggat actgctcaac aatctggtat tgaaatgatg 840
gcctacgatt tccatactga acaacctatt aagggtgccc atatctatca tttgggtcat 900
atcttgcata accacccaga tcatatctgc agagatatct tgaagagaat tgctgaagct 960
atgtccccaa agtccagatt attgattttc gatgctgtta ttccagccag aactgaacca 1020
ggtaagtata tgaatggtta cttgatcgat atcgttggtt tggctactgg tggtaaagaa 1080
agaactgaaa aagaaaccgc tgctttgttg gatgctgaag gtttggaatt tgttagagtt 1140
tggcatggtc aagctggttg gcaaggtgtt attgaagcaa gattgaaaag aggttaa 1197
<210> 2
<211> 398
<212> PRT
<213> Lasiodiplodia theobromae
<400> 2
Met Arg Ser Tyr Ser Leu Asp Ile Val Pro Lys Ile Glu Ala Ile Leu
1 5 10 15
Ala Asp Ala Lys Gln Leu Glu Asp Glu Asp Leu Ser Ala Arg Leu Gly
20 25 30
Leu Met Thr Gln Val Asp Arg Leu Tyr Gln Asp Leu Glu Ser Pro Ile
35 40 45
Glu Leu Phe Phe Arg Gln Trp Thr Asp Val Thr Val Tyr Ser Cys Val
50 55 60
Asn Val Ala Ile Lys Leu Gly Ile Phe Glu Lys Met Lys Ala Lys Glu
65 70 75 80
Asn Ile Ser Ala Gln Glu Leu Ala Asp Leu Val Gly Val Asp Leu Asp
85 90 95
Val Ile Val Arg Val Met Arg Val Leu Val Ala Ala Gly Ile Val Ala
100 105 110
Ser Pro Val Glu Glu Thr Tyr Ser His Thr Pro Lys Ser Leu Ile Phe
115 120 125
Val Arg Gly Lys Gly Thr Ala Val Gly Ser Phe Glu Leu Ile Ala Leu
130 135 140
Tyr Ala Lys Ser Tyr Leu Ser Leu Pro Glu Tyr Leu Lys Thr Arg Pro
145 150 155 160
Ser Ala Asp Leu Tyr Asp Val Cys Lys Thr Pro Tyr Ala Tyr Glu Tyr
165 170 175
Gly Ile Glu Gly Lys Pro Phe Tyr Glu Ala Leu Ser Ser Val Pro Gln
180 185 190
His Leu Asp Ile Phe Asn Arg Ala Met Asp Glu Pro Thr Thr Glu Cys
195 200 205
Gly Ile Phe Pro Trp Ser Ser Leu Lys Glu Glu Val Gln Ala Glu Pro
210 215 220
Asp Arg Ala Phe Ile Val Asp Ile Gly Gly Gly Ser Gly Lys Val Leu
225 230 235 240
His Phe Ala Arg Ala Glu Thr Gly Asp Val Phe Gly Thr Ser Ala Arg
245 250 255
Leu Ile Leu Gln Glu Arg Pro Asp Ala Leu Ala Gln Leu Asp Thr Ala
260 265 270
Gln Gln Ser Gly Ile Glu Met Met Ala Tyr Asp Phe His Thr Glu Gln
275 280 285
Pro Ile Lys Gly Ala His Ile Tyr His Leu Gly His Ile Leu His Asn
290 295 300
His Pro Asp His Ile Cys Arg Asp Ile Leu Lys Arg Ile Ala Glu Ala
305 310 315 320
Met Ser Pro Lys Ser Arg Leu Leu Ile Phe Asp Ala Val Ile Pro Ala
325 330 335
Arg Thr Glu Pro Gly Lys Tyr Met Asn Gly Tyr Leu Ile Asp Ile Val
340 345 350
Gly Leu Ala Thr Gly Gly Lys Glu Arg Thr Glu Lys Glu Thr Ala Ala
355 360 365
Leu Leu Asp Ala Glu Gly Leu Glu Phe Val Arg Val Trp His Gly Gln
370 375 380
Ala Gly Trp Gln Gly Val Ile Glu Ala Arg Leu Lys Arg Gly
385 390 395
<210> 3
<211> 23
<212> DNA
<213> Lasiodiplodia theobromae
<400> 3
gatctgggtg gttatgcaag ata 23
<210> 4
<211> 21
<212> DNA
<213> Lasiodiplodia theobromae
<400> 4
accatctgct gacttgtacg a 21

Claims (6)

1. a kind of zearalenone poison-removing method, it is characterised in that: utilize the oxygen methyl modification enzyme Lt- of Lasiodiplodia theobromae 3 hydroxyls (- OH) of zearalenone are replaced with oxygen methyl group (- OCH by OMT3)。
2. a kind of zearalenone poison-removing method according to claim 1, it is characterised in that: by the gene of Lt-OMT with PRS425 carrier connection building artificial recombination plasmid 92-Lt-OMT-YEp-Leu, by artificial recombination plasmid 92-Lt-OMT-YEp- Leu is transferred in competence yeast cells, and yeast cells is placed in Leu auxotroph culture medium and is cultivated, in Leu auxotrophy Zearalenone is added in type culture medium and realizes co-cultivation, the Lt-OMT of yeast cells synthesis converts zearalenone to 3-OCH3- ZEN, reaction equation are as follows:
3. a kind of zearalenone poison-removing method according to claim 2, it is characterised in that: specific steps are as follows:
(1) gene cloning:
Normal chain and negative strand primer are designed according to the nucleotide sequence of Lt-OMT, by the method for PCR, obtains target gene Lt- OMT；
(2) building of recombinant plasmid:
1. utilizing restriction enzyme NdeI, PmeI digestion Lt-OMT PCR product and pRS425 carrier, pass through T4DNA ligase Lt-OMT genetic fragment after digestion is connect construction recombination plasmid 92-Lt-OMT-Yep-Leu with pRS425 carrier after digestion；
It is expressed 2. recombinant plasmid 92-Lt-OMT-Yep-Leu is imported in competent E.coli DH5 α cell, alkaline lysis mentions Plasmid is taken, it is whether complete using agarose electrophoresis verifying purpose gene is carried out after NdeI and PmeI digested plasmid；
(3) yeast conversion:
The 92-Lt-OMT-Yep-Leu recombinant plasmid of building is imported in competence yeast cells BJ5464-NpgA；
(4) zearalenone and yeast transformant co-culture:
10mg zearalenone is placed in the auxotroph-Leu culture solution containing yeast transformant and cultivates 48h；It is described Yeast transformant is the yeast cells containing 1 μ g 92-Lt-OMT-Yep-Leu recombinant plasmid；
(5) tunning extracts:
(6) converted product is purified:
Each fraction is collected with each component of the mobile phase separation crude extract of different proportion by silica gel column chromatography；By each fraction Thin-layer chromatography contact plate is carried out, target compound is screened；
(7) converted product structural analysis.
4. a kind of zearalenone poison-removing method according to claim 3, it is characterised in that: the specific step of step (5) Suddenly are as follows:
1. fermentation liquid 1580g is centrifuged 5min, supernatant, sediment are collected respectively；
2. 1. middle supernatant is mixed with isometric ethyl acetate, poured into separatory funnel after mixing well, stratification, point It Shou Ji not supernatant, sediment；
3. the sediment of step 2. is uniformly mixed with isometric ethyl acetate, stratification, supernatant, precipitating are collected respectively Object；
4. the sediment of step 3. is uniformly mixed with isometric ethyl acetate, stratification, supernatant, precipitating are collected respectively Object；
5. 2. 3. 4. supernatant mixes and be transferred to Rotary Evaporators by step, it is concentrated into paste；
6. 3 times of volumes methanols and ultrasound 15min are added in 1. sediment, pour into separatory funnel, stratification is collected respectively Supernatant, sediment；
7. by step, 6. sediment is uniformly mixed with isometric ethyl acetate, is extracted three times repeatedly, is merged supernatant three times；
8. by step, 7. supernatant is transferred to Rotary Evaporators, is concentrated into paste；
9. 5. 8. concentrate that step is obtained chromatography methanol redissolves, after 0.22 μm of membrane filtration, 13400g centrifugation 10min, supernatant are fitted into spare in brown liquid-phase inlet bottle.
5. a kind of zearalenone poison-removing method according to claim 3, it is characterised in that: the specific step of step (7) Suddenly are as follows:
1. tunning disappears through high-efficient liquid phase chromatogram technique analysis, the zearalenone substrate peak of 4.790min, 4.175min generates a new peak；
2. collecting converted product fraction, i.e. compound at 4.175min, rotation steaming method is precipitated and weighs to white crystalline powder；
3. the molecular weight through mass spectrometer analysis noval chemical compound is 333.8；
4. being obtained through NMR analysis, new compound structure 3-OCH3-ZEN；
5. analysis show that the enzyme is 98% to the transformation efficiency of zearalenone in yeast fermentation system BJ5464-NpgA.
6. a kind of zearalenone poison-removing method according to claim 3, it is characterised in that: the nucleotides sequence of Lt-OMT Column are as shown in SEQ ID NO.1, and amino acid sequence is as shown in SEQ ID NO.2.
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