CN109439640A - Fumonisin degrading enzyme FumDSB and its gene and application - Google Patents
Fumonisin degrading enzyme FumDSB and its gene and application Download PDFInfo
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Abstract
The invention belongs to agricultural biological technical fields, and in particular to fumonisin degrading enzyme FumDSB and its gene and application.The present invention provides a kind of source Sphingomonadales Bacterium fumonisin degrading enzyme FumDSB, amino acid sequence is as shown in SEQ ID NO.1, and the present invention provides the encoding genes for encoding above-mentioned detoxication enzyme.Fumonisin degrading enzyme of the invention can be applied to that agricultural, feed and food etc. are industrial, reduce harm of the fumonisin to animal and human health.
Description
Technical field
The invention belongs to agricultural biological technical fields, and in particular to fumonisin degrading enzyme FumDSB and its gene and answer
With.
Background technique
Fumonisin (Fumonisin, FB) is one that Fusarium (Fusarium spp.) generates under given conditions
A little water-soluble secondary metabolites.Fumonisin forms di esters compound by the pure and mild tricarballylic acid of more hydrogen, in structure
Chief active functional group is primary amine, tricarboxylic acids, hydroxyl and aliphatic skeleton, these are closely related with their poisonous effect.It grinds
Study carefully and shows that fumonisin can cause rat kidney neoplasms and liver cancer, horse white matter of brain malacosis (ELEM), pig pulmonary edema (PPE) etc., and
It is closely related with the morbidity of human esophagus cancer.Up to the present, it was found that the analog of 28 kinds of fumonisins is classified as four
Class, i.e. FA, FB, FC and FP, in B series, most often it is found out that fumonisins B1 (FB1), B2 (FB2) and B3 (FB3), wherein
The amount of FB1 amount in nature accounts about 70%-80% or so, and toxicity is most strong, pollutes most extensive.Applied Physics, chemistry
Method can remove fumonisin, chemical method limitation is bigger, although the content of FB1 can be reduced, but can introduce new
Chemical substance, increase security risk, furthermore European Union be also forbidden to use chemical method eliminate mycotoxin.Physical method can remove
A certain amount of FB1, but requirement condition is high, and malleable food quality and flavor.Biological degradation method degrades FB1 reaction mildly, specially
One property is strong, and specificity is high, and application prospect is boundless.
The pollution of mycotoxin is all increasingly sharpening in the world, causes the waste of valuable grain resource, and
There is carcinogenicity to people.Therefore, the enzyme preparation for researching and developing degradative fungi toxin, can be very good to contain in contaminated cereal crops
Toxin generation, retrieve economic losses.
Summary of the invention
In order to which using biological degradation method degradation fumonisin, the present invention provides a kind of fumonisin degrading enzyme FumDSB
And its gene and application.
The purpose of the present invention is to provide a kind of fumonisin degrading enzyme FumDSB.
A further object of the present invention is to provide the genes of fumonisin degrading enzyme FumDSB a kind of.
A further object of the present invention is to provide a kind of, and the recombinant expression containing fumonisin degrading enzyme FumDSB gene carries
Body.
A further object of the present invention is to provide a kind of recombinant bacterial strains containing fumonisin degrading enzyme FumDSB gene.
A further object of the present invention is to provide the methods for preparing above-mentioned fumonisin degrading enzyme FumDSB.
A further object of the present invention is to provide the applications of above-mentioned fumonisin degrading enzyme FumDSB.
The fumonisin degrading enzyme FumDSB of specific embodiment according to the present invention, amino acid sequence such as SEQ ID
Shown in NO.1:
MTLASLLLLAPAVAHAGPSVVTTDAGRLRGAVEGELDVFRGVPFAAAPIGQLRWREPQRIAPWTDIRDA
SKFAPACMQSGVSIPGEPAPQISEDCLYLNIWAPRHSGRAKLPVMIFIHGGGWQNGATALPLYWGDRLAQQGAVVVS
VSYRLGALGFLAHPELTAESPHHTSGNYGLLDQIAALNWVQRNIAAFGGDPANVTLFGQSAGSSSIAILMASPLAKG
LFHRVIGQSGGFFEPLQLAPHYELALAEKQGVAFAHSLETSTLADLRSLPPQALLTKQAASVSHPVIEPWLLPRTPF
EVFSVGQQHGADILVGYNAEEGRAFFDASSVTAANFGEQLRAELGDLPPAIMAAYPFASDVEAGQARVALERDLRFG
WNMWTWAKLHAATGKNAVHAYYFTHKPPFPSDSVRRNWQASHFAELWYMFDHLGQEDWQWTKFDRQIARTMSRYWVN
FARNGNPNGRGLPHWPAYRTDQPLVLQIGQPITPTPEPNTGSIGVIDAVFSAVRGDT
Wherein, which encodes 511 amino acid and a terminator codon, and preceding 16 amino acids are signal peptides, because
This, the theoretical molecular weight of mature FB1 degrading enzyme FumDSB is 54kDa.
Mature its amino acid sequence of fumonisin degrading enzyme FumDSB of the invention is as shown in SEQ ID NO.2:
PSVVTTDAGRLRGAVEGELDVFRGVPFAAAPIGQLRWREPQRIAPWTDIRDASKFAPACMQSGVSIPGE
PAPQISEDCLYLNIWAPRHSGRAKLPVMIFIHGGGWQNGATALPLYWGDRLAQQGAVVVSVSYRLGALGFLAHPELT
AESPHHTSGNYGLLDQIAALNWVQRNIAAFGGDPANVTLFGQSAGSSSIAILMASPLAKGLFHRVIGQSGGFFEPLQ
LAPHYELALAEKQGVAFAHSLETSTLADLRSLPPQALLTKQAASVSHPVIEPWLLPRTPFEVFSVGQQHGADILVGY
NAEEGRAFFDASSVTAANFGEQLRAELGDLPPAIMAAYPFASDVEAGQARVALERDLRFGWNMWTWAKLHAATGKNA
VHAYYFTHKPPFPSDSVRRNWQASHFAELWYMFDHLGQEDWQWTKFDRQIARTMSRYWVNFARNGNPNGRGLPHWPA
YRTDQPLVLQIGQPITPTPEPNTGSIGVIDAVFSAVRGDT
The present invention provides the genomes for the gene for encoding above-mentioned fumonisin degrading enzyme FumDSB, and nucleotide sequence is such as
Shown in SEQ ID NO.3:
atgacgctggcttccttgcttctcctcgctccggcagttgcacatgccggacccagtgtcgtcaccac
cgacgccggccggcttcgtggagcggtcgaaggcgagcttgacgtgttcagaggcgtaccttttgcggcagcgccc
attggtcaacttcgttggcgcgagcctcagcggatcgcgccttggaccgatatacgggatgccagcaaatttgccc
cggcatgcatgcaaagcggtgtttcaatcccgggtgagccagcgccgcagatcagcgaagattgcctatacctcaa
tatttgggcgccgcggcattctggtcgggcgaagctgcccgtaatgattttcattcatggcggcggttggcagaat
ggtgcgaccgcgctgccactctattggggcgatcggttggcgcagcaaggggctgtcgtcgtttcggtgagctacc
ggcttggtgcgcttggcttcctcgcccatcccgaacttaccgccgaatcaccacatcacacgtcagggaactacgg
cctgctcgatcaaatcgccgccctcaattgggtgcagcgcaacatcgcggcgtttgggggtgacccggcaaatgtt
actctatttggtcagtcggcgggttcgagttcgatcgctatcctgatggcttcaccgttggccaagggcctgtttc
atcgcgtcatcggccaaagtggcggttttttcgagccgctacaactggccccgcattacgaactcgcgctcgctga
aaaacagggagttgcctttgcacattccctagaaacttccacgcttgctgatctccggtcgctccccccgcaggcc
ttgttgacgaaacaggccgccagcgtgtcgcatcctgtaatcgaaccatggctattgccgcgaaccccgtttgagg
ttttctcggtagggcagcaacacggtgcagacattctcgtcggatacaatgccgaagaaggccgagcattctttga
cgcttcgtcggtcactgctgcgaacttcggtgaacagcttcgcgccgaattgggcgacctgcctcctgcaatcatg
gcggcctacccttttgccagcgatgtcgaggcggggcaggcgcgcgttgcgctggagcgggatctgcgtttcggct
ggaacatgtggacctgggcaaagctccacgccgcaaccggaaaaaacgcggtccatgcctattacttcacccacaa
accaccgttcccaagcgattcagttcgaagaaattggcaggcaagccacttcgccgaactgtggtacatgtttgac
catcttggccaagaagattggcaatggacaaagtttgatcgtcaaatcgctcgcaccatgtcgcgttactgggtaa
attttgcccggaacggaaatcccaacgggcgaggtcttccgcactggcctgcctatcgtaccgaccaaccgcttgt
gttgcagattggccaaccgataacacccacgccggagcccaataccgggtctattggagtgatcgacgcggttttt
tcagcagtacgaggcgacacctga
The present invention obtains fumonisin degrading enzyme FumDSB gene, DNA complete sequence point by full genome synthetic method
Analysis the result shows that, degrading enzyme FumDSB structural gene overall length 1536bp.
Mature fumonisin degrading enzyme FumDPS encoding gene of the invention, the sequence of nucleotide such as SEQ ID
Shown in NO.4:
ggacccagtgtcgtcaccaccgacgccggccggcttcgtggagcggtcgaaggcgagcttgacgtgtt
cagaggcgtaccttttgcggcagcgcccattggtcaacttcgttggcgcgagcctcagcggatcgcgccttggacc
gatatacgggatgccagcaaatttgccccggcatgcatgcaaagcggtgtttcaatcccgggtgagccagcgccgc
agatcagcgaagattgcctatacctcaatatttgggcgccgcggcattctggtcgggcgaagctgcccgtaatgat
tttcattcatggcggcggttggcagaatggtgcgaccgcgctgccactctattggggcgatcggttggcgcagcaa
ggggctgtcgtcgtttcggtgagctaccggcttggtgcgcttggcttcctcgcccatcccgaacttaccgccgaat
caccacatcacacgtcagggaactacggcctgctcgatcaaatcgccgccctcaattgggtgcagcgcaacatcgc
ggcgtttgggggtgacccggcaaatgttactctatttggtcagtcggcgggttcgagttcgatcgctatcctgatg
gcttcaccgttggccaagggcctgtttcatcgcgtcatcggccaaagtggcggttttttcgagccgctacaactgg
ccccgcattacgaactcgcgctcgctgaaaaacagggagttgcctttgcacattccctagaaacttccacgcttgc
tgatctccggtcgctccccccgcaggccttgttgacgaaacaggccgccagcgtgtcgcatcctgtaatcgaacca
tggctattgccgcgaaccccgtttgaggttttctcggtagggcagcaacacggtgcagacattctcgtcggataca
atgccgaagaaggccgagcattctttgacgcttcgtcggtcactgctgcgaacttcggtgaacagcttcgcgccga
attgggcgacctgcctcctgcaatcatggcggcctacccttttgccagcgatgtcgaggcggggcaggcgcgcgtt
gcgctggagcgggatctgcgtttcggctggaacatgtggacctgggcaaagctccacgccgcaaccggaaaaaacg
cggtccatgcctattacttcacccacaaaccaccgttcccaagcgattcagttcgaagaaattggcaggcaagcca
cttcgccgaactgtggtacatgtttgaccatcttggccaagaagattggcaatggacaaagtttgatcgtcaaatc
gctcgcaccatgtcgcgttactgggtaaattttgcccggaacggaaatcccaacgggcgaggtcttccgcactggc
ctgcctatcgtaccgaccaaccgcttgtgttgcagattggccaaccgataacacccacgccggagcccaataccgg
gtctattggagtgatcgacgcggttttttcagcagtacgaggcgacacc
Fumonisin degrading enzyme FumDSB sequence and its amino acid sequence are subjected to BLAST comparison, ammonia in GenBank
Base acid sequence is 33% with sphingol box bacterium MTA144 Amino acid sequence identity is derived from, and illustrates fumonisin drop of the invention
Solution enzyme FumDSB is a kind of new FB1 detoxication enzyme.
The present invention also provides the recombinant vector comprising above-mentioned fumonisin degrading enzyme FumDSB, preferred recombinant vector life
Entitled pET28a-FumDSB.Fumonisin degrading enzyme FumDSB gene of the invention is inserted into expression vector suitably to limit
Between property restriction enzyme site, make its nucleotide sequence is operable to be linked to the expression control sequence.Most as of the invention one
Detoxifying gene of the invention is preferably inserted into EcoR I and Xhol on plasmid pET28a limitation by preferred embodiment
Property restriction enzyme site between, so that the nucleotide sequence is located at the downstream of T7 promoter and is regulated and controled by it, obtain recombination large intestine expression matter
Grain pET28a-FumDSB.
The present invention also provides the recombinant bacterial strain comprising above-mentioned degrading enzyme FumDSB, preferred strain is Escherichia coli.
The present invention also provides the methods of fumonisin degrading enzyme FumDSB comprising following steps:
(1) host cell is converted with the recombinant vector of the gene comprising encoding fumonisin degrading enzyme FumDSB, obtains weight
Group bacterial strain；
(2) recombinant bacterial strain, induction fumonisin degrading enzyme FumDSB expression are cultivated；
(3) the fumonisin degrading enzyme FumDSB of acquisition is isolated and purified.
The present invention also provides the applications of above-mentioned fumonisin degrading enzyme FumDSB, especially answering in degradation fumonisin B1
With.
30 DEG C of optimum temperature of fumonisin degrading enzyme FumDSB of the invention, optimal pH 6.0, in pH5.5-9 range
Inside still keep 60% or more activity.Under optimum temperature and optimal pH, fumonisin degrading enzyme FumDSB of the invention is to FB1
Degradation rate be 100%.
The fumonisin B1 fumonisin degrading enzyme of good properties can be obtained using method of the invention, which can answer
It is industrial for agricultural, feed and food etc., reduce harm of the fumonisin B1 to animal and human health.
Detailed description of the invention
The SDS-PAGE that Fig. 1 is fumonisin degrading enzyme FumDSB purifies figure, wherein M: albumen marker；1: volt horse poison
Plain degrading enzyme FumDSB；
Fig. 2 shows the optimum temperature situation of fumonisin degrading enzyme FumDSB；
Fig. 3 shows the optimal pH situation of fumonisin degrading enzyme FumDSB.
Specific embodiment
Test material and reagent
1, bacterial strain and carrier: coli expression carrier pET28a and coli strain Bl21 (DE3)；
2, culture medium:
Escherichia coli culture medium LB (1% peptone, 0.5% yeast extract, 1%NaCl, pH7.0).
Embodiment 1 obtains fumonisin degrading enzyme FumDSB gene
It is obtained using artificial chemistry synthetic method and is dropped from Sphingomonadales Bacterium fumonisin
The genetic fragment of enzyme FumDSB is solved, and introduces restriction enzyme site EcoR I, in 3 ' end introducing restriction enzyme site Xho I at 5 ' ends.
The preparation of 2 fumonisin degrading enzyme FumDSB of embodiment
Expression vector pET28a is subjected to double digestion (EcoR I+Xho I), while by encoding mature fumonisin degrading enzyme
Gene FumDSB double digestion (EcoR I+Xho I), cut out coding degrading enzyme genetic fragment and expression vector pET28a connect
It connects, obtains the recombinant plasmid pET28a-FumDSB containing degrading enzyme FumDSB and convert to e. coli bl21 (DE3), weighed
Group coli strain BL21 (DE3)/FumDSB.
The recombinant expression carrier of the fumonisin degrading enzyme gene containing signal peptide sequence is constructed in the same way.
BL21 (DE3) bacterial strain containing recombinant plasmid is taken, is inoculated in 100mL LB culture solution, 37 DEG C of 220rpm oscillation trainings
After supporting 2-3h, OD600When for 0.6-0.8, final concentration of 1mM IPTG, 25 DEG C of induction 20h is added, induction terminates, and 4 DEG C of centrifugations are received
Collect thallus.By ultrasonication, collect supernatant, after ni-sepharose purification, SDS-PAGE the result shows that, recombinate detoxication enzyme in Escherichia coli
In expressed, as shown in Figure 1, swimming lane 1 is fumonisin degrading enzyme FumDSB after purification.
The property of 3 fumonisin degrading enzyme FumDSB of embodiment measures
The enzyme activity of high performance liquid chromatography detection fumonisin degrading enzyme, the specific method is as follows:
(1) FB1 standard reserving solution: claiming 1mg to mark product, is dissolved with 10ml acetonitrile and water (1:1), and being configured to concentration is 100ug/
The titer of mL, -20 DEG C of preservations.
(2) preparation of sample: taking purified fumonisin degrading enzyme enzyme solution 900ul, and the FB1 standard storage of 100ul is added
Standby liquid makes the final concentration of 10ug/ml of FB1,37 DEG C, 220rpm, is protected from light culture 20min.
(3) sample derivatization: taking sample to be tested 100ul, and 50% acetonitrile water of 400ul is added, and the derivative liquid of 500ul OPA mixes
Sample introduction in even 30s, derivative 2min, filter membrane are to be measured.It is compared by the peak figure of the mark product with FB1, to determine that fumonisin is degraded
The enzymatic activity of enzyme FumDSB.
1. measuring the optimum temperature of fumonisin degrading enzyme FumDSB
Under citrate-phosphate disodium hydrogen buffer (pH7.0) buffer solution system and different temperatures, using FB1 as substrate, eventually
Concentration is 10 μ g/ml, takes 100 μ L substrates and the enzyme solution of 900 μ L is added, react 20min, then boiling water boiling 10min, allows enzyme to lose
It is living.Film is crossed after being cooled to room temperature, and is detected to HPLC.
As shown in Fig. 2, the optimum temperature of fumonisin degrading enzyme FumDSB of the invention is 30 DEG C.
2. measuring the optimal pH of fumonisin degrading enzyme FumDSB
Select different buffer solution: 100mM citrate-phosphate disodium hydrogen (pH 3.0-8.0), the Tris-Hcl of 100mM
(pH8-9), 100mM glycine-NaOH (pH 9.0-12.0) carries out enzymatic reaction under above-mentioned different pH buffer, to survey
Its fixed optimal pH.
As shown in figure 3, the optimal pH of fumonisin degrading enzyme FumDSB of the invention is 6.0.
Meanwhile the present invention is also measured the temperature stability and pH stability of fumonisin degrading enzyme FumDSB, knot
Fruit shows that fumonisin degrading enzyme FumDSB has preferable temperature stability and pH stability.
Sequence table
<110>University Of Science and Technology Of Tianjin
<120>fumonisin degrading enzyme FumDSB and its gene and application
<160> 4
<170> SIPOSequenceListing 1.0
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<212> PRT
<213>sphingolipid monad (Sphingomonadales Bacterium)
<400> 1
Met Thr Leu Ala Ser Leu Leu Leu Leu Ala Pro Ala Val Ala His Ala
1 5 10 15
Gly Pro Ser Val Val Thr Thr Asp Ala Gly Arg Leu Arg Gly Ala Val
20 25 30
Glu Gly Glu Leu Asp Val Phe Arg Gly Val Pro Phe Ala Ala Ala Pro
35 40 45
Ile Gly Gln Leu Arg Trp Arg Glu Pro Gln Arg Ile Ala Pro Trp Thr
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Asp Ile Arg Asp Ala Ser Lys Phe Ala Pro Ala Cys Met Gln Ser Gly
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Val Ser Ile Pro Gly Glu Pro Ala Pro Gln Ile Ser Glu Asp Cys Leu
85 90 95
Tyr Leu Asn Ile Trp Ala Pro Arg His Ser Gly Arg Ala Lys Leu Pro
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Val Met Ile Phe Ile His Gly Gly Gly Trp Gln Asn Gly Ala Thr Ala
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Leu Pro Leu Tyr Trp Gly Asp Arg Leu Ala Gln Gln Gly Ala Val Val
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Val Ser Val Ser Tyr Arg Leu Gly Ala Leu Gly Phe Leu Ala His Pro
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Glu Leu Thr Ala Glu Ser Pro His His Thr Ser Gly Asn Tyr Gly Leu
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Leu Asp Gln Ile Ala Ala Leu Asn Trp Val Gln Arg Asn Ile Ala Ala
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Phe Gly Gly Asp Pro Ala Asn Val Thr Leu Phe Gly Gln Ser Ala Gly
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Ser Ser Ser Ile Ala Ile Leu Met Ala Ser Pro Leu Ala Lys Gly Leu
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Phe His Arg Val Ile Gly Gln Ser Gly Gly Phe Phe Glu Pro Leu Gln
225 230 235 240
Leu Ala Pro His Tyr Glu Leu Ala Leu Ala Glu Lys Gln Gly Val Ala
245 250 255
Phe Ala His Ser Leu Glu Thr Ser Thr Leu Ala Asp Leu Arg Ser Leu
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Pro Pro Gln Ala Leu Leu Thr Lys Gln Ala Ala Ser Val Ser His Pro
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Val Ile Glu Pro Trp Leu Leu Pro Arg Thr Pro Phe Glu Val Phe Ser
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Val Gly Gln Gln His Gly Ala Asp Ile Leu Val Gly Tyr Asn Ala Glu
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Glu Gly Arg Ala Phe Phe Asp Ala Ser Ser Val Thr Ala Ala Asn Phe
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Gly Glu Gln Leu Arg Ala Glu Leu Gly Asp Leu Pro Pro Ala Ile Met
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Ala Ala Tyr Pro Phe Ala Ser Asp Val Glu Ala Gly Gln Ala Arg Val
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Ala Leu Glu Arg Asp Leu Arg Phe Gly Trp Asn Met Trp Thr Trp Ala
370 375 380
Lys Leu His Ala Ala Thr Gly Lys Asn Ala Val His Ala Tyr Tyr Phe
385 390 395 400
Thr His Lys Pro Pro Phe Pro Ser Asp Ser Val Arg Arg Asn Trp Gln
405 410 415
Ala Ser His Phe Ala Glu Leu Trp Tyr Met Phe Asp His Leu Gly Gln
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Glu Asp Trp Gln Trp Thr Lys Phe Asp Arg Gln Ile Ala Arg Thr Met
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Ile Gly Val Ile Asp Ala Val Phe Ser Ala Val Arg Gly Asp Thr
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Gly Gln Leu Arg Trp Arg Glu Pro Gln Arg Ile Ala Pro Trp Thr Asp
35 40 45
Ile Arg Asp Ala Ser Lys Phe Ala Pro Ala Cys Met Gln Ser Gly Val
50 55 60
Ser Ile Pro Gly Glu Pro Ala Pro Gln Ile Ser Glu Asp Cys Leu Tyr
65 70 75 80
Leu Asn Ile Trp Ala Pro Arg His Ser Gly Arg Ala Lys Leu Pro Val
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Met Ile Phe Ile His Gly Gly Gly Trp Gln Asn Gly Ala Thr Ala Leu
100 105 110
Pro Leu Tyr Trp Gly Asp Arg Leu Ala Gln Gln Gly Ala Val Val Val
115 120 125
Ser Val Ser Tyr Arg Leu Gly Ala Leu Gly Phe Leu Ala His Pro Glu
130 135 140
Leu Thr Ala Glu Ser Pro His His Thr Ser Gly Asn Tyr Gly Leu Leu
145 150 155 160
Asp Gln Ile Ala Ala Leu Asn Trp Val Gln Arg Asn Ile Ala Ala Phe
165 170 175
Gly Gly Asp Pro Ala Asn Val Thr Leu Phe Gly Gln Ser Ala Gly Ser
180 185 190
Ser Ser Ile Ala Ile Leu Met Ala Ser Pro Leu Ala Lys Gly Leu Phe
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His Arg Val Ile Gly Gln Ser Gly Gly Phe Phe Glu Pro Leu Gln Leu
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Ala Pro His Tyr Glu Leu Ala Leu Ala Glu Lys Gln Gly Val Ala Phe
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Ala His Ser Leu Glu Thr Ser Thr Leu Ala Asp Leu Arg Ser Leu Pro
245 250 255
Pro Gln Ala Leu Leu Thr Lys Gln Ala Ala Ser Val Ser His Pro Val
260 265 270
Ile Glu Pro Trp Leu Leu Pro Arg Thr Pro Phe Glu Val Phe Ser Val
275 280 285
Gly Gln Gln His Gly Ala Asp Ile Leu Val Gly Tyr Asn Ala Glu Glu
290 295 300
Gly Arg Ala Phe Phe Asp Ala Ser Ser Val Thr Ala Ala Asn Phe Gly
305 310 315 320
Glu Gln Leu Arg Ala Glu Leu Gly Asp Leu Pro Pro Ala Ile Met Ala
325 330 335
Ala Tyr Pro Phe Ala Ser Asp Val Glu Ala Gly Gln Ala Arg Val Ala
340 345 350
Leu Glu Arg Asp Leu Arg Phe Gly Trp Asn Met Trp Thr Trp Ala Lys
355 360 365
Leu His Ala Ala Thr Gly Lys Asn Ala Val His Ala Tyr Tyr Phe Thr
370 375 380
His Lys Pro Pro Phe Pro Ser Asp Ser Val Arg Arg Asn Trp Gln Ala
385 390 395 400
Ser His Phe Ala Glu Leu Trp Tyr Met Phe Asp His Leu Gly Gln Glu
405 410 415
Asp Trp Gln Trp Thr Lys Phe Asp Arg Gln Ile Ala Arg Thr Met Ser
420 425 430
Arg Tyr Trp Val Asn Phe Ala Arg Asn Gly Asn Pro Asn Gly Arg Gly
435 440 445
Leu Pro His Trp Pro Ala Tyr Arg Thr Asp Gln Pro Leu Val Leu Gln
450 455 460
Ile Gly Gln Pro Ile Thr Pro Thr Pro Glu Pro Asn Thr Gly Ser Ile
465 470 475 480
Gly Val Ile Asp Ala Val Phe Ser Ala Val Arg Gly Asp Thr
485 490
<210> 3
<211> 1536
<212> DNA
<213>sphingolipid monad (Sphingomonadales Bacterium)
<400> 3
atgacgctgg cttccttgct tctcctcgct ccggcagttg cacatgccgg acccagtgtc 60
gtcaccaccg acgccggccg gcttcgtgga gcggtcgaag gcgagcttga cgtgttcaga 120
ggcgtacctt ttgcggcagc gcccattggt caacttcgtt ggcgcgagcc tcagcggatc 180
gcgccttgga ccgatatacg ggatgccagc aaatttgccc cggcatgcat gcaaagcggt 240
gtttcaatcc cgggtgagcc agcgccgcag atcagcgaag attgcctata cctcaatatt 300
tgggcgccgc ggcattctgg tcgggcgaag ctgcccgtaa tgattttcat tcatggcggc 360
ggttggcaga atggtgcgac cgcgctgcca ctctattggg gcgatcggtt ggcgcagcaa 420
ggggctgtcg tcgtttcggt gagctaccgg cttggtgcgc ttggcttcct cgcccatccc 480
gaacttaccg ccgaatcacc acatcacacg tcagggaact acggcctgct cgatcaaatc 540
gccgccctca attgggtgca gcgcaacatc gcggcgtttg ggggtgaccc ggcaaatgtt 600
actctatttg gtcagtcggc gggttcgagt tcgatcgcta tcctgatggc ttcaccgttg 660
gccaagggcc tgtttcatcg cgtcatcggc caaagtggcg gttttttcga gccgctacaa 720
ctggccccgc attacgaact cgcgctcgct gaaaaacagg gagttgcctt tgcacattcc 780
ctagaaactt ccacgcttgc tgatctccgg tcgctccccc cgcaggcctt gttgacgaaa 840
caggccgcca gcgtgtcgca tcctgtaatc gaaccatggc tattgccgcg aaccccgttt 900
gaggttttct cggtagggca gcaacacggt gcagacattc tcgtcggata caatgccgaa 960
gaaggccgag cattctttga cgcttcgtcg gtcactgctg cgaacttcgg tgaacagctt 1020
cgcgccgaat tgggcgacct gcctcctgca atcatggcgg cctacccttt tgccagcgat 1080
gtcgaggcgg ggcaggcgcg cgttgcgctg gagcgggatc tgcgtttcgg ctggaacatg 1140
tggacctggg caaagctcca cgccgcaacc ggaaaaaacg cggtccatgc ctattacttc 1200
acccacaaac caccgttccc aagcgattca gttcgaagaa attggcaggc aagccacttc 1260
gccgaactgt ggtacatgtt tgaccatctt ggccaagaag attggcaatg gacaaagttt 1320
gatcgtcaaa tcgctcgcac catgtcgcgt tactgggtaa attttgcccg gaacggaaat 1380
cccaacgggc gaggtcttcc gcactggcct gcctatcgta ccgaccaacc gcttgtgttg 1440
cagattggcc aaccgataac acccacgccg gagcccaata ccgggtctat tggagtgatc 1500
gacgcggttt tttcagcagt acgaggcgac acctga 1536
<210> 4
<211> 1485
<212> DNA
<213>sphingolipid monad (Sphingomonadales Bacterium)
<400> 4
ggacccagtg tcgtcaccac cgacgccggc cggcttcgtg gagcggtcga aggcgagctt 60
gacgtgttca gaggcgtacc ttttgcggca gcgcccattg gtcaacttcg ttggcgcgag 120
cctcagcgga tcgcgccttg gaccgatata cgggatgcca gcaaatttgc cccggcatgc 180
atgcaaagcg gtgtttcaat cccgggtgag ccagcgccgc agatcagcga agattgccta 240
tacctcaata tttgggcgcc gcggcattct ggtcgggcga agctgcccgt aatgattttc 300
attcatggcg gcggttggca gaatggtgcg accgcgctgc cactctattg gggcgatcgg 360
ttggcgcagc aaggggctgt cgtcgtttcg gtgagctacc ggcttggtgc gcttggcttc 420
ctcgcccatc ccgaacttac cgccgaatca ccacatcaca cgtcagggaa ctacggcctg 480
ctcgatcaaa tcgccgccct caattgggtg cagcgcaaca tcgcggcgtt tgggggtgac 540
ccggcaaatg ttactctatt tggtcagtcg gcgggttcga gttcgatcgc tatcctgatg 600
gcttcaccgt tggccaaggg cctgtttcat cgcgtcatcg gccaaagtgg cggttttttc 660
gagccgctac aactggcccc gcattacgaa ctcgcgctcg ctgaaaaaca gggagttgcc 720
tttgcacatt ccctagaaac ttccacgctt gctgatctcc ggtcgctccc cccgcaggcc 780
ttgttgacga aacaggccgc cagcgtgtcg catcctgtaa tcgaaccatg gctattgccg 840
cgaaccccgt ttgaggtttt ctcggtaggg cagcaacacg gtgcagacat tctcgtcgga 900
tacaatgccg aagaaggccg agcattcttt gacgcttcgt cggtcactgc tgcgaacttc 960
ggtgaacagc ttcgcgccga attgggcgac ctgcctcctg caatcatggc ggcctaccct 1020
tttgccagcg atgtcgaggc ggggcaggcg cgcgttgcgc tggagcggga tctgcgtttc 1080
ggctggaaca tgtggacctg ggcaaagctc cacgccgcaa ccggaaaaaa cgcggtccat 1140
gcctattact tcacccacaa accaccgttc ccaagcgatt cagttcgaag aaattggcag 1200
gcaagccact tcgccgaact gtggtacatg tttgaccatc ttggccaaga agattggcaa 1260
tggacaaagt ttgatcgtca aatcgctcgc accatgtcgc gttactgggt aaattttgcc 1320
cggaacggaa atcccaacgg gcgaggtctt ccgcactggc ctgcctatcg taccgaccaa 1380
ccgcttgtgt tgcagattgg ccaaccgata acacccacgc cggagcccaa taccgggtct 1440
attggagtga tcgacgcggt tttttcagca gtacgaggcg acacc 1485
Claims (10)
1. fumonisin degrading enzyme FumDSB, which is characterized in that its amino acid sequence such as SEQ ID NO.1 or SEQ ID NO.2
It is shown.
2. fumonisin degrading enzyme FumDSB gene, which is characterized in that encode fumonisin degrading enzyme described in claim 1
FumDSB。
3. fumonisin degrading enzyme FumDSB gene according to claim 2, which is characterized in that its nucleotide sequence is such as
Shown in SEQ ID NO.3 or SEQ ID NO.4.
4. including the recombinant vector of fumonisin degrading enzyme FumDSB gene as claimed in claim 2.
5. including recombinant vector pET28a (+)-FumDSB of fumonisin degrading enzyme FumDSB gene as claimed in claim 2.
6. including the recombinant bacterial strain of fumonisin degrading enzyme FumDSB gene as claimed in claim 2.
7. recombinant bacterial strain according to claim 6, which is characterized in that the bacterial strain is Escherichia coli.
8. the method for preparing fumonisin degrading enzyme FumDSB described in claim 1, it is characterised in that: the method includes with
Lower step:
(1) host is converted with the recombinant vector of the gene comprising encoding fumonisin degrading enzyme FumDSB described in claim 1
Cell obtains recombinant bacterial strain；
(2) recombinant bacterial strain, induction fumonisin degrading enzyme FumDSB expression are cultivated；
(3) the fumonisin degrading enzyme FumDSB of acquisition is isolated and purified.
9. the application of fumonisin degrading enzyme FumDSB described in claim 1.
10. application of the fumonisin degrading enzyme FumDSB described in claim 1 in terms of the fumonisin B1 that degrades.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111607575A (en) * | 2020-04-07 | 2020-09-01 | 天津科技大学 | Transaminase PHTA, preparation method and application |
| CN112239755A (en) * | 2020-09-22 | 2021-01-19 | 天津科技大学 | Fumonisin degrading enzyme FumDSS and gene and application thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1196601A1 (en) * | 1999-07-14 | 2002-04-17 | Pioneer Hi-Bred International Inc. | Compositions and methods for fumonisin detoxification |
| CN102159706A (en) * | 2008-09-18 | 2011-08-17 | 爱尔伯股份公司 | Method for production of additive for enzymatic decomposition of mycotoxins, additive, and use thereof |
| WO2012032472A2 (en) * | 2010-09-06 | 2012-03-15 | Danisco A/S | Additive |
| CN106661541A (en) * | 2014-04-17 | 2017-05-10 | 贝林格尔·英格海姆Rcv两合公司 | Recombinant host cell engineered to overexpress helper proteins |
-
2018
- 2018-10-09 CN CN201811171697.3A patent/CN109439640B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1196601A1 (en) * | 1999-07-14 | 2002-04-17 | Pioneer Hi-Bred International Inc. | Compositions and methods for fumonisin detoxification |
| CN102159706A (en) * | 2008-09-18 | 2011-08-17 | 爱尔伯股份公司 | Method for production of additive for enzymatic decomposition of mycotoxins, additive, and use thereof |
| WO2012032472A2 (en) * | 2010-09-06 | 2012-03-15 | Danisco A/S | Additive |
| CN106661541A (en) * | 2014-04-17 | 2017-05-10 | 贝林格尔·英格海姆Rcv两合公司 | Recombinant host cell engineered to overexpress helper proteins |
Non-Patent Citations (5)
| Title |
|---|
| BAGNOUD,A.等: ""carboxylesterase [Sphingomonadales bacterium BRH_c42]"", 《GENBANK DATABASE》 * |
| DORIS HARTINGER等: "Enzyme characteristics of aminotransferase FumI of Sphingopyxis sp. MTA144 for deamination of hydrolyzed fumonisin B1", 《APPL MICROBIOL BIOTECHNOL》 * |
| STEFAN HEINL等: "Degradation of fumonisin B1 by the consecutive action of two bacterial enzymes", 《JOURNAL OF BIOTECHNOLOGY》 * |
| ZHONGYUAN LI等: "Biological detoxification of fumonisin by a novel carboxylesterase from Sphingomonadales bacterium and its biochemical characterization", 《INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES》 * |
| 陈亭亭等: "基于羧酸酯酶降解伏马毒素研究", 《试验研究》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111607575A (en) * | 2020-04-07 | 2020-09-01 | 天津科技大学 | Transaminase PHTA, preparation method and application |
| CN112239755A (en) * | 2020-09-22 | 2021-01-19 | 天津科技大学 | Fumonisin degrading enzyme FumDSS and gene and application thereof |
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| CN109439640B (en) | 2021-07-20 |
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