CN108277210A - Mould ketenes hydrolase ZEN214 and encoding gene and application - Google Patents
Mould ketenes hydrolase ZEN214 and encoding gene and application Download PDFInfo
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- CN108277210A CN108277210A CN201711364503.7A CN201711364503A CN108277210A CN 108277210 A CN108277210 A CN 108277210A CN 201711364503 A CN201711364503 A CN 201711364503A CN 108277210 A CN108277210 A CN 108277210A
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- Prior art keywords
- zen214
- hydrolase
- zeranol
- application
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
- C12N9/18—Carboxylic ester hydrolases (3.1.1)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
Abstract
The present invention relates to feeding enzyme fields, more particularly to zeranol hydrolase ZEN214 and encoding gene and application, its amino acid sequence is as shown in SEQ ID NO.1, the enzyme being capable of hydrolysed corn zeranol, toxin is removed, the application in animal goes mouldy feed and feedstuff has vast potential for future development.
Description
Technical field
The present invention relates to feeding enzyme fields, and in particular to mould ketenes hydrolase ZEN214 and encoding gene and application.
Background technology
Zearalenone (Zearalenone, ZEN) be distributed in the world it is most a wide range of it is a kind of generated by sickle-like bacteria it is true
Verticillium toxin has generation in the cereal and agricultural and sideline product on the ground such as Asia, Europe and America.ZEN has oestrogen-like hormone property, again
F-2 toxin, entitled 6- (10- hydroxyls -6- oxygroups-undecenyl) β of chemistry-thunder is claimed to lock acid lactone, molecular formula is
C18H22O5, molecular mass 318, fusing point are 165 DEG C, are relatively stablized to heat, and 120 DEG C of heating 4h are not degraded.ZEN has very strong
Genotoxicity, can with estrogen receptor generate competitive binding then show estrogenic activity.Estrogen receptor and ZEN
It changes in conjunction with rear receptor conformation, is transferred in nucleus and is further combined with chromatin, adjust genetic transcription and protein
This two big process is synthesized, therefore the division of cell and growth is made to be affected.ZEN mainly pollutes the farmings such as corn, wheat, barley
Object and feed can cause the livestock or poultries such as pig, chicken precocity, cyclostage disorderly, massive losses brought to kind of an aquaculture.ZEN
Also there is strong carcinogenicity, cause the incidence such as breast cancer, the cancer of the esophagus to increase, become the reason of cancer morbidity rises successively now
One of.The detection method of ZEN is more perfect at present, but the problem of related ZEN conversions, degradation etc. is extremely urgent still outstanding
And it is pending.
ZEN pollution ranges are extensive, and the extent of injury is serious, therefore, effectively control and solve its pollution to grain and feed,
There is very important meaning safely to improving breeding performonce fo animals and improving human food.ZEN biodegradation methods in recent years
Increasingly it is taken seriously.The enzyme and ZEN that the biodegradation method of ZEN is primarily referred to as generating when microorganism, plant or its metabolism are sent out
It is raw to act on and ZEN molecular structure Poisoning groups is made to be destroyed to generate the process of nontoxic metabolite.Due to microorganism and
The method of its bio-enzyme degradation ZEN can thoroughly remove toxin, meanwhile, the specificity of this method is strong, it is pollution-free to feed, will not
Feed nutritive value is influenced, therefore, this method is before the application during animal goes mouldy feed and feedstuff has wide development
Scape.
Invention content
The object of the present invention is to provide a kind of zearalenone hydrolase ZEN214.
It is a further object of the present invention to provide the gene zen214 for encoding above-mentioned zearalenone hydrolase ZEN214.
It is a further object of the present invention to provide the recombinant vectors comprising said gene.
It is a further object of the present invention to provide the recombinant bacterial strains comprising said gene.
It is a further object of the present invention to provide a kind of methods preparing above-mentioned zearalenone hydrolase ZEN214.
It is a further object of the present invention to provide the applications of above-mentioned zearalenone hydrolase ZEN214.
The present invention has synthesized a kind of new zearalenone hydrolase ZEN214, and constructing being capable of high efficient expression this enzyme
Recombinant escherichia coli strain.
The present invention provides a kind of zearalenone hydrolase ZEN214, amino acid sequence is as follows：
SEQ ID NO.1：
MVFERLKSTVTTKDGIEWYYEQEGVGPHVVLIPDGLGECQMMDKPMSLIASAGFTVTTFDMPGMSRSSN
APVESYKGVTGHKLATQLDTLMEHLEIAKASFWGCSAGALAVLGLCAHYPSRVRNAMPHEAPLHLMDQLKDLPSLDD
ETLIASMAAISKASCRDDEAWNSLGPEVHQRLRRNFIRWAHGYISDLTLSPPIETENLRKRPISWTVGADSPMFMFF
SNVVTATKADIPIKTLPGSHFPYVSHPEQMSKHIVEATRSHLP
Wherein, which includes 266 amino acid, does not predict signal peptide sequence, which is
29.3kDa。
The present invention provides the gene zen214 for encoding above-mentioned zearalenone hydrolase ZEN214, encoding gene warps
Sequence is as follows after codon optimization：
SEQ ID NO.2：
atggttttcgaaagattgaagtctactgttactactaaagatggtattgagtggtactatgaacaagagggtgttgg
tccacatgttgttttgattcctgatggtttgggtgaatgtcaaatgatggataagccaatgtctttgattgcttctg
ctggttttactgttactactttcgatatgccaggaatgtctagatcttctaacgctcctgttgaatcttacaagggt
gttactggtcataaattggctactcaattggatactttgatggaacacttggagattgctaaagcttctttttgggg
ttgttctgctggtgctttggctgttttgggtttgtgtgctcactatccatctagagttagaaatgctatgccacatg
aagctcctttgcacttgatggatcaattgaaggatttgccttctttggatgatgagactttgattgcttctatggct
gctatttctaaagcttcttgtagagatgatgaagcttggaattctttgggtccagaggttcatcaaagattgagaag
aaacttcatcagatgggctcacggttacatttctgatttgactttgtctccacctattgaaactgagaacttgagaa
agagaccaatttcttggactgttggtgctgattctcctatgttcatgtttttctctaacgttgttactgctactaag
gctgatatcccaattaaaactttgcctggtagtcatttcccatatgtttctcaccctgaacaaatgtctaaacatat
tgttgaggctactagatctcacttgccttaa
The present invention provides the recombinant vector for including above-mentioned zeranol hydrolase gene zen214, preferably pet30a-
zen214.The zeranol hydrolase gene of the present invention is inserted between suitable restriction enzyme cleavage sites of the expression vector, is made
Its nucleotide sequence is operable to be linked to the expression control sequence.It is excellent as the most preferred embodiment of the present invention
Be selected as the restricted digestion positions the Nde I and Not I zeranol hydrolase gene of the present invention being inserted on plasmid pet30a
Between point, so that the nucleotide sequence is located at the downstream of T7 promoters and is regulated and controled by it, obtain expression of recombinant e. coli plasmid
pet30a-zen214。
The present invention also provides the recombinant bacterial strain for including above-mentioned zeranol hydrolase gene zen214, the preferably described bacterial strains
For e. coli bl21/zen214.
The present invention also provides a kind of methods preparing zeranol hydrolase ZEN214, include the following steps：
1) host cell is converted with above-mentioned recombinant vector, obtains recombinant bacterial strain；
2) recombinant bacterial strain is cultivated, induction recombination zeranol hydrolyzes expression of enzymes；
3) it recycles and purifies expressed zeranol hydrolase ZEN214 albumen.
Specific implementation mode according to the present invention, in expression in escherichia coli zeranol hydrolase.It is red mould in order to measure
The property of ketenes hydrolase passes through such as ammonium sulfate precipitation, the straightforward procedure purifying zeranol hydrolysis of dialysis, ultrafiltration and chromatography
Enzyme.By simply purifying, the purity of zeranol hydrolase is enough to study the research of zymologic property.
The present invention also provides the applications of above-mentioned zeranol hydrolase ZEN214, including biomass energy, food industry
With the application of feed industry etc..
The zeranol hydrolase ZEN214 of group expression has wide in range pH ranges, is all had preferably in pH 5.0-9.0
Hydrolysis ability, optimum temperature range are 35-40 degrees Celsius.
Description of the drawings
Fig. 1 shows the SDS-PAGE analysis results of zeranol hydrolase.
Fig. 2 shows zeranol hydrolase zearalenone substrate HPLC analysis results.
Specific implementation mode
Test material and reagent
1, bacterial strain and carrier：The encoding gene (SEQ ID NO.2) of the zeranol hydrolase ZEN214 of the present invention is in gold
Si Rui companies synthesize.
2, enzyme and other biochemical reagents：Restriction endonuclease is purchased from TaKaRa companies, and ligase is purchased from Invitrogen companies.Its
It is all domestic reagent (can be commercially available from common biochemical Reagent Company).
3, Escherichia coli culture medium LB (1% peptone, 0.5% yeast extract, 1%NaCl, pH are natural).
Explanation：Do not make the experimental methods of molecular biology illustrated, equal reference in following embodiment《Molecular Cloning: A Laboratory
Guide》Listed specific method carries out in one book of (third edition) J. Pehanorm Brookers, or according to kit and product description
It carries out.
Embodiment 1 recombinates the structure of zeranol hydrolase ZEN214 expression vectors
Using the method for overlap-PCR, expression vector pet30a and zeranol hydrolase are compiled through two-step pcr method
Code gene zen214 connections obtain recombinant plasmid pet30a-zen214 and convert e. coli bl21, obtain recombination bacillus coli
Bacterial strain BL21/zen214.
First step PCR the primers are as follows：
214-30a F:5'-TATGCACCATCATCATCATCATATGGTTTTCGAAAGATTG-3'
214-30a R:5'-AGTGGTGGTGGTGGTGGTGTTAAGGCAAGTGAGATCTAGT-3'
30a-214F:5'-GCCTTAACACCACCACCACCACCACTGAGATCCG-3'
30a-214R:5'-TCGAAAACCATATGATGATGATGATGGTGCATAT-3'
Wherein 214-30a F and 214-30a R are used to expand the segment of zen214, and 30a-214F and 30a-214R is for expanding
Increase the carrier segments of pet30a.
PCR reaction systems are as follows：
PCR reaction conditions are：95℃5min；94 DEG C of 30sec, 60 DEG C of 30sec, 72 DEG C of 2.5min, 30 cycles；72℃,
10min；4℃,hold.
After amplification, PCR product is subjected to nucleic acid electrophoresis detection, stripe size is respectively 801bp and 5500bp, by it
It is separately recovered purifying, carries out the second wheel PCR, which need not be added primer, two sections of products of the first step PCR primer and mould each other
Plate it is amplifiable go out recombination segment, control addition genetic fragment and carrier segments molar ratio be 1：1, concentration is in 50ng/ μ l.
PCR reaction systems are as follows：
PCR reaction conditions are：95℃5min；94 DEG C of 30sec, 62 DEG C of 30sec, 72 DEG C of 3.5min, 30 cycles；72℃,
10min；4℃,hold.
After amplification, verifies its PCR product through nucleic acid electrophoresis and be 6300bp or be gathered in point sample in polymer form
Kong Zhong, you can directly by 5 μ LPCR products conversion Escherichia coli Trans, I competence.
After sequencing is correct, recombinant plasmid pet30a-zen214 conversions E. coli competent BL21 is subjected to induction table
It reaches.3-5 positive transformant of picking is seeded in the LB liquid medium that 30ml contains 100 μ g/ml ampicillins respectively, and 37
DEG C, 250rpm cultivate 6 hours, this is preculture；This preculture is forwarded to the LB that 300ml contains 100 μ g/ml ampicillins
In fluid nutrient medium, 37 DEG C, 250rpm cultivate 2-3 hours, this OD600 is about 0.4.1M IPTG liquid storages are added to this culture
In object, so that IPTG final concentrations is reached 0.06mM, while cultivation temperature is reduced to 25 DEG C, maintain the rotating speed of shaking table constant, continues
Induction 8 hours.4000g is centrifuged 15 minutes, is abandoned supernatant and is collected bacterium.With 25ml Tris buffer solutions (20mM Tris-HCl,
150mMNaCl, pH7.5) gravity treatment thalline, with ultrasonic disruption microorganism, 12000g is centrifuged 15 minutes, and weight is contained in supernatant
The zeranol hydrolase protein of group.By supernatant and 1ml Ni-NTA metal chelate chromatographies, washs 3 times and contain recombinant protein
Ni-NTA metal chelate chromatography agar, then with containing various concentration (100mM-400mM) imidazoles elution buffer (20mM Tris-
HCl, 150mMNaCl, pH7.5) recombinant protein is eluted from agar.SDS-PAGE verifies the purity of albumen, merges most
The albumen of pure part, and recombinant protein dialyses with more to Tris buffer solutions (20mM Tris-HCl, 150mMNaCl, pH7.5)
Change buffer solution.
Embodiment 2 recombinates the activity analysis of zeranol hydrolase
The ZEN214 of the zeranol hydrolase of codon optimization, expressing quantity ratio improve about before being not optimised
2.2 times, production cost can be significantly reduced, expression and protein SDS-PAGE analysis result after purification are as shown in Figure 1, red mould alkene
Ketone hydrolase zearalenone substrate HPLC analysis as shown in Fig. 2,
Reaction system is：
The disodium hydrogen phosphate citrate buffer solution of the pH6.0 of 800ul, in addition 100ul is dissolved in the red mould of the 0.5g/L of DMSO
Ketenes toxin (is purchased from sigma companies), then the appropriate diluted enzyme solution of addition 100ul processes, 37 degrees Celsius of warm bath hours, then
The DMSO of 2 times of volumes of addition terminates reaction, while acutely after concussion, after absorption sample segment crosses miillpore filter, the efficient liquid of loading
Facies analysis (HPLC) is compareed and is inactivated within 5 minutes in boiling water bath for appropriate diluted enzyme solution.
The zeranol hydrolase ZEN214 of group expression has wide in range pH ranges, is all had preferably in pH 5.0-9.0
Hydrolysis ability, optimum temperature range are 35-40 DEG C.
Sequence table
<110>Institute of Feeds,China Academy of Agriculture Sciences
<120>Mould ketenes hydrolase ZEN214 and encoding gene and application
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 266
<212> PRT
<213>Artificial sequence (Artificial Sequence)
<400> 1
Met Val Phe Glu Arg Leu Lys Ser Thr Val Thr Thr Lys Asp Gly Ile
1 5 10 15
Glu Trp Tyr Tyr Glu Gln Glu Gly Val Gly Pro His Val Val Leu Ile
20 25 30
Pro Asp Gly Leu Gly Glu Cys Gln Met Met Asp Lys Pro Met Ser Leu
35 40 45
Ile Ala Ser Ala Gly Phe Thr Val Thr Thr Phe Asp Met Pro Gly Met
50 55 60
Ser Arg Ser Ser Asn Ala Pro Val Glu Ser Tyr Lys Gly Val Thr Gly
65 70 75 80
His Lys Leu Ala Thr Gln Leu Asp Thr Leu Met Glu His Leu Glu Ile
85 90 95
Ala Lys Ala Ser Phe Trp Gly Cys Ser Ala Gly Ala Leu Ala Val Leu
100 105 110
Gly Leu Cys Ala His Tyr Pro Ser Arg Val Arg Asn Ala Met Pro His
115 120 125
Glu Ala Pro Leu His Leu Met Asp Gln Leu Lys Asp Leu Pro Ser Leu
130 135 140
Asp Asp Glu Thr Leu Ile Ala Ser Met Ala Ala Ile Ser Lys Ala Ser
145 150 155 160
Cys Arg Asp Asp Glu Ala Trp Asn Ser Leu Gly Pro Glu Val His Gln
165 170 175
Arg Leu Arg Arg Asn Phe Ile Arg Trp Ala His Gly Tyr Ile Ser Asp
180 185 190
Leu Thr Leu Ser Pro Pro Ile Glu Thr Glu Asn Leu Arg Lys Arg Pro
195 200 205
Ile Ser Trp Thr Val Gly Ala Asp Ser Pro Met Phe Met Phe Phe Ser
210 215 220
Asn Val Val Thr Ala Thr Lys Ala Asp Ile Pro Ile Lys Thr Leu Pro
225 230 235 240
Gly Ser His Phe Pro Tyr Val Ser His Pro Glu Gln Met Ser Lys His
245 250 255
Ile Val Glu Ala Thr Arg Ser His Leu Pro
260 265
<210> 2
<211> 801
<212> DNA
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 2
atggttttcg aaagattgaa gtctactgtt actactaaag atggtattga gtggtactat 60
gaacaagagg gtgttggtcc acatgttgtt ttgattcctg atggtttggg tgaatgtcaa 120
atgatggata agccaatgtc tttgattgct tctgctggtt ttactgttac tactttcgat 180
atgccaggaa tgtctagatc ttctaacgct cctgttgaat cttacaaggg tgttactggt 240
cataaattgg ctactcaatt ggatactttg atggaacact tggagattgc taaagcttct 300
ttttggggtt gttctgctgg tgctttggct gttttgggtt tgtgtgctca ctatccatct 360
agagttagaa atgctatgcc acatgaagct cctttgcact tgatggatca attgaaggat 420
ttgccttctt tggatgatga gactttgatt gcttctatgg ctgctatttc taaagcttct 480
tgtagagatg atgaagcttg gaattctttg ggtccagagg ttcatcaaag attgagaaga 540
aacttcatca gatgggctca cggttacatt tctgatttga ctttgtctcc acctattgaa 600
actgagaact tgagaaagag accaatttct tggactgttg gtgctgattc tcctatgttc 660
atgtttttct ctaacgttgt tactgctact aaggctgata tcccaattaa aactttgcct 720
ggtagtcatt tcccatatgt ttctcaccct gaacaaatgt ctaaacatat tgttgaggct 780
actagatctc acttgcctta a 801
Claims (9)
1. zearalenone hydrolase ZEN214, which is characterized in that its amino acid sequence is as shown in SEQ ID NO.1.
2. zearalenone hydrolase gene, which is characterized in that encode zearalenone hydrolase described in claim 1
ZEN214。
3. zearalenone hydrolase gene according to claim 2, which is characterized in that its nucleotide sequence such as SEQ
Shown in ID NO.2.
4. including the recombinant vector of zearalenone hydrolase gene described in claim 2.
5. including the recombinant vector pet30a-zen214 of zearalenone hydrolase gene described in claim 2.
6. including the recombinant cell of zearalenone hydrolase gene described in claim 2.
7. recombinant cell according to claim 6, which is characterized in that the recombinant cell is Bacillus coli cells or yeast
Cell.
8. the application of zearalenone hydrolase ZEN214 described in claim 1.
9. the application of zearalenone hydrolase gene described in claim 2.
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| CN201711364503.7A CN108277210B (en) | 2017-12-18 | 2017-12-18 | Mycetone hydrolase ZEN214, and coding gene and application thereof |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110527677A (en) * | 2019-09-02 | 2019-12-03 | 中国农业科学院饲料研究所 | Zearalenone hydrolyzes enzyme mutant ZHDM2 and its encoding gene and application |
| CN110564707A (en) * | 2019-09-02 | 2019-12-13 | 中国农业科学院饲料研究所 | Zearalenone hydrolase mutant ZHDM1 and coding gene and application thereof |
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| CN107217046A (en) * | 2017-06-28 | 2017-09-29 | 国家粮食局科学研究院 | A kind of zearalenone toxin degradation enzyme ZENdease N1 and its encoding gene and application |
| CN107446902A (en) * | 2017-06-28 | 2017-12-08 | 国家粮食局科学研究院 | A kind of zearalenone toxin degradation enzyme ZENdease N2 and its encoding gene and application |
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| CN107217046A (en) * | 2017-06-28 | 2017-09-29 | 国家粮食局科学研究院 | A kind of zearalenone toxin degradation enzyme ZENdease N1 and its encoding gene and application |
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| CN110527677A (en) * | 2019-09-02 | 2019-12-03 | 中国农业科学院饲料研究所 | Zearalenone hydrolyzes enzyme mutant ZHDM2 and its encoding gene and application |
| CN110564707A (en) * | 2019-09-02 | 2019-12-13 | 中国农业科学院饲料研究所 | Zearalenone hydrolase mutant ZHDM1 and coding gene and application thereof |
| CN110527677B (en) * | 2019-09-02 | 2021-07-20 | 中国农业科学院北京畜牧兽医研究所 | Zearalenone hydrolase mutant ZHDM2 and coding gene and application thereof |
| CN110564707B (en) * | 2019-09-02 | 2021-11-05 | 中国农业科学院北京畜牧兽医研究所 | Zearalenone hydrolase mutant ZHDM1 and coding gene and application thereof |
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