阅读说明：本技术 染料脱色过氧化物酶StDyP用于同时降解黄曲霉毒素和玉米赤霉烯酮的应用 (Application of dye decolorization peroxidase StDyP in simultaneous degradation of aflatoxin and zearalenone ) 是由 秦星 罗会颖 王晓璐 涂涛 苏小运 张�杰 黄火清 柏映国 王苑 王亚茹 姚斌 于 2021-08-05 设计创作，主要内容包括：本发明涉及农业生物技术领域,具体涉及染料脱色过氧化物酶StDyP用于同时降解黄曲霉毒素和玉米赤霉烯酮的应用。所述染料脱色过氧化物酶StDyP的氨基酸序列如SEQ ID NO:1所示。本发明获得了染料脱色过氧化物酶StDyP,将其应用在霉菌毒素脱毒方面,其能够有效降解黄曲霉毒素B1和玉米赤霉烯酮。(The invention relates to the technical field of agricultural biology, in particular to a dye decoloration peroxidase St DyP for the simultaneous degradation of aflatoxins and zearalenone. The dye decolorizing peroxidase St DyP is shown in SEQ ID NO 1. The invention obtains the dye decoloration peroxidase St DyP, when the compound is applied to the detoxification of mycotoxin, the compound can effectively degrade aflatoxin B1 and zearalenone.)

1. Dye decolorizing peroxidaseStDyP use for the simultaneous degradation of aflatoxins and zearalenone, characterized in that the dye decolorizing peroxidaseStDyP is shown in SEQ ID NO 1.

2. A method for simultaneously degrading aflatoxin and zearalenone in feed, which comprises decolorizing peroxidase with dyeStDyP degradation of aflatoxin and zearalenone, wherein the dye decolorization peroxidaseStDyP is shown in SEQ ID NO 1.

3. The method for simultaneously degrading aflatoxin and zearalenone in feed according to claim 2, which comprises constructing a recombinant DNA encoding said dye decolorizing peroxidaseStDyP in the presence of a recombinant vector.

4. The method for simultaneously degrading aflatoxins and zearalenone in a feed according to claim 3, which comprises the step of expressing the obtained recombinant vector in a host cell.

5. The method for simultaneously degrading aflatoxin and zearalenone in feed according to claim 3, wherein said dye decolorizing peroxidase is encodedStDyP is shown in SEQ ID NO. 2.

6. The method for simultaneously degrading aflatoxin and zearalenone in a feed according to claim 4, wherein the host cell is Escherichia coli.

7. Dye decolorizing peroxidaseStDyP use for degrading aflatoxins, wherein the dye decolorizing peroxidaseStDyP is shown in SEQ ID NO 1.

8. Dye decolorizing peroxidaseStDyP use for degrading zearalenone, wherein the dye decolorizes peroxidaseStDyP is shown in SEQ ID NO 1.

Technical Field

The invention relates to the technical field of agricultural biology, in particular to a dye decoloration peroxidaseStDyP for the simultaneous degradation of aflatoxins and zearalenone.

Background

Mycotoxins are toxic secondary metabolites produced by fungi of aspergillus, penicillium, fusarium and the like, have the characteristics of carcinogenicity, teratogenicity, immunotoxicity, neurotoxicity, reproductive and developmental toxicity and the like, and can enter human and animal bodies through food, feed or food, so that the health of the human and animal is seriously harmed. More than 400 fungal toxins are reported at present, and according to the source of the toxins, the fungal toxins can be divided into fusarium fungal toxin, aspergillus fungal toxin, penicillium fungal toxin and ergot fungal toxin, and the fungal toxins from different sources can be further subdivided according to toxin structures, wherein the common fungal toxins in the feed comprise aflatoxin, zearalenone, trichothecene toxins (deoxynivalenol), ochratoxin and fumonisins. The common mycotoxins not only reduce the feed quality, but also indirectly influence the production performance of livestock and poultry, have adverse effects on the tissue function and health of animals, and bring potential food safety problems.

The quality safety of the feed is the key point for guaranteeing the healthy development of the animal husbandry, and the problem of mycotoxin pollution in the feed is a key problem to be solved urgently. Mycotoxins are ubiquitous in domestic feeds and raw materials, with aflatoxins, zearalenone, vomitoxin, and fumonisins being the major mycotoxins. It is noteworthy that the contamination of the mycotoxins in the feed and raw materials is often not of a single toxin, but of a plurality of mycotoxins simultaneously. Related research reports indicate that the synergistic effect of various mycotoxins has a greater toxic effect on animal health and growth performance than a single mycotoxin. Most of the reported degrading enzymes only aim at a single toxin molecule, and cannot effectively degrade different types of toxins, so that the development of the degrading enzymes capable of simultaneously degrading a plurality of toxins to be low-toxic or non-toxic is urgently needed.

Disclosure of Invention

The invention aims to provide a dye decolorizing peroxidaseStDyP for the simultaneous degradation of aflatoxins and zearalenone.

It is a further object of the present invention to provide a method for degrading aflatoxins and zearalenone.

Dye-decolorizing peroxidase according to the present inventionStDyP use for the simultaneous degradation of aflatoxins and zearalenone, wherein the dye decolorization peroxidaseStDyP is shown in SEQ ID NO 1.

Dye-decolorizing peroxidase according to the present inventionStDyP use for the simultaneous degradation of aflatoxins and zearalenone, wherein the above dye decolorizing peroxidase is encodedStDyP is shown in SEQ ID NO. 2.

Wherein the enzyme comprises 425 amino acids, mature dye decolorizing peroxidaseStDyP has a theoretical molecular weight of 45.5 kDa.

The method for simultaneously degrading aflatoxin and zearalenone in feed comprises the step of decolorizing peroxidase by using dyeStDyP degradation of aflatoxin and zearalenone, wherein the dye decolorization peroxidaseStDyP is shown in SEQ ID NO 1.

The method for simultaneously degrading aflatoxin and zearalenone in feed according to the invention comprises constructing a protein containing a coding sequence encoding the dyeMaterial decolorizing peroxidaseStDyP in the presence of a recombinant vector.

The method for simultaneously degrading aflatoxin and zearalenone in a feed according to the invention comprises the step of expressing the obtained recombinant vector in a host cell.

The method for simultaneously degrading aflatoxin and zearalenone in feed according to the invention, wherein the dye decolorizing peroxidase is encodedStDyP is shown in SEQ ID NO. 2.

The present invention provides a dye decolorizing peroxidaseStDyP use for degrading aflatoxins, wherein the dye decolorizing peroxidaseStDyP is shown in SEQ ID NO 1.

The present invention provides a dye decolorizing peroxidaseStDyP use for degrading zearalenone, wherein the dye decolorizes peroxidaseStDyP is shown in SEQ ID NO 1.

According to a specific embodiment of the present invention, the decolorizing peroxidase containing the above dyeStDyP the recombinant vector preferably is pCold I-StDyP are provided. The dye decolorizing peroxidase gene of the present invention is inserted between suitable restriction sites of an expression vector to operably link its nucleotide sequence with an expression regulatory sequence. As a most preferred embodiment of the present invention, the dye-decolorizing peroxidase gene of the present invention isStDyP insertion into plasmid pCold INdeI-XbaI between restriction sites, so that the nucleotide sequence is locatedcspThe downstream of the A promoter and the lac operon is regulated and controlled to obtain recombinant Escherichia coli pG-Tf2/BL21 plasmid pCold I-StDyP。

According to a specific embodiment of the present invention, the dye decolorizing peroxidase gene as described above is containedStDyP, preferably recombinant Escherichia coli pG-Tf2/BL21 strainE. coli pG-Tf2/BL21/StDyP。

By comparison of NCBI databases withStDyP the most consistent enzyme is derived from StreptomycesThe peroxidase EfeB of unknown species, but its biochemical properties have not been reported. Peroxidase with verified biochemical function and the method of the present inventionStDyP the enzyme has a maximum identity of only 43.1%.

The invention obtains the dye decoloration peroxidaseStDyP, when the compound is applied to the detoxification of mycotoxin, the compound can effectively degrade aflatoxin B1 and zearalenone.

Drawings

FIG. 1 shows recombinant dye decolorizing peroxidaseStDyP degradation rate of aflatoxin B1 and zearalenone;

FIG. 2 shows recombinant dye decolorizing peroxidaseStDyP HPLC analysis result of aflatoxin B1 degradation;

FIG. 3 shows recombinant dye decolorizing peroxidaseStDyP HPLC analysis of zearalenone degradation.

Detailed Description

Test materials and reagents

1. Genes and vectors: an escherichia coli expression vector pCold I and a strain escherichia coli pG-Tf2/BL 21;

2. enzymes and other biochemical reagents: endonuclease, recombinase, aflatoxin B1 and zearalenone.

3. Culture medium: coli medium LB (1% peptone, 0.5% yeast extract, 1% NaCl, pH 7.0).

Example 1 dye decolorizing peroxidaseStDyP cloning of the Gene encoding

The target gene of the invention is derived from streptomycesStreptomyces thermocarboxydusDye decolorizing peroxidase ofStDyP。

Sequence specific primers:

StDyP-F: SEQ ID NO:3；StDyP-R: SEQ ID NO:4。

by streptomyceteStreptomyces thermocarboxydusThe genomic DNA of (3) was used as a template for PCR amplification. Electrophoresing on 1% agarose gel, cutting to obtain target fragment, recovering the fragment, and mixing withNdeI-XbaThe pCold I vector with double enzyme digestion is connected by a homologous recombination method to transform Trans I gramCloning host, sequencing and verifying to obtain the dye decolorizing peroxidaseStDyP encodes a gene.

Example 2 recombinant dye decolorizing peroxidaseStDyP preparation

The obtained gene containing dye decolorization peroxidaseStDyP recombinant E.coli expression plasmid pCold I-StDyP transformation of Escherichia coli pG-Tf2/BL21 to obtain recombinant Escherichia coli pG-Tf2/BL21StDyP。

GetE. coli pG-Tf2/BL21/StDyP strain was inoculated into 50 mL LB medium, cultured with shaking at 37 ℃ and 200 rpm for 12 hours, then transferred to 300 mL LB medium at a ratio of 2%, cultured with shaking at 37 ℃ and 200 rpm for about 4 hours (OD 600. apprxeq.0.6), then added with 1 mM inducer IPTG and 10. mu.M Hemin solution to the final concentration, cultured with induction at 16 ℃ and 200 rpm for 12 hours, and then centrifuged to collect the cells. The cells were resuspended in equilibration buffer (20 mM pH 7.4 Na)₂HPO₄-NaH₂PO₄500 mM NaCl). The cells were lysed by ultrasonication. Centrifuging to remove broken thallus fragment, purifying with Ni affinity chromatography column, collecting electrophoretically pure eluate, and dialyzing to protein stock solution (20 mM pH 7.4 Na)₂HPO₄-NaH₂PO₄) In (1).

Example 3 recombinant dye decolorizing peroxidaseStDyP degradation of aflatoxin B1

Dissolving aflatoxin B1 into dimethyl sulfoxide to prepare a mother solution with the concentration of 100 mg/L, and reacting according to the following reaction system: mu.L malonic acid buffer (0.2M, pH 5.0), 20. mu.L aflatoxin B1 solution (100 mg/L), 20. mu.L manganese sulfate (10 mM), 100. mu.L dye-decolorizing peroxidase (500U/L), 5. mu.L hydrogen peroxide (4 mM). The system without the dye decolorizing peroxidase was used as a control, and the reaction system was set to 3 replicates. The reaction is carried out at 30 ℃, methanol with the same volume is added after 72 hours to stop the reaction, and the degradation rate of the aflatoxin B1 is analyzed by High Performance Liquid Chromatography (HPLC). The liquid chromatogram is Shimadzu LC-20A high performance liquid chromatography, and the chromatographic separation column is Waters Xbridge C18 column (4.6 × 150 mm, 5 μm), mobile phase A (water), and mobile phase B (methanol); eluting for 20 minutes under the condition of equal gradient elution and 45 percent B; and (4) detecting by using a fluorescence detector, wherein the excitation wavelength is 360 nm, and the emission wavelength is 440 nm.

As a result, as shown in FIGS. 1 and 2, it can be seen that a part of aflatoxin was degraded, and the degradation rate was 21.03%.

Example 4 recombinant dye decolorizing peroxidaseStDyP degrading zearalenone

Dissolving zearalenone in dimethyl sulfoxide to prepare a mother solution with the concentration of 100 mg/L, and carrying out the following reaction system: 55. mu.L of malonic acid buffer (0.2M, pH 5.0), 20. mu.L of zearalenone solution (100 mg/L), 20. mu.L of manganese sulfate (10 mM), 100. mu.L of dye-decolorizing peroxidase (500U/L), 5. mu.L of hydrogen peroxide (4 mM). The system without the dye decolorizing peroxidase was used as a control, and the reaction system was set to 3 replicates. The reaction is carried out at 30 ℃, methanol with the same volume is added after 72 hours to stop the reaction, and the degradation rate of the zearalenone is analyzed by High Performance Liquid Chromatography (HPLC). The liquid chromatogram is Shimadzu LC-20A high performance liquid chromatography, and the chromatographic separation column is Waters Xbridge C18 column (4.6 × 150 mm, 5 μm), mobile phase A (water), and mobile phase B (acetonitrile); eluting for 20 minutes under the condition of equal gradient elution and 45 percent B; the fluorescence detector is adopted for detection, the excitation wavelength is 274 nm, and the emission wavelength is 440 nm.

The results are shown in fig. 1 and fig. 3, and it can be seen that part of zearalenone had been degraded, and the degradation rate was 45.37%.

Sequence listing

<110> Beijing animal husbandry and veterinary institute of Chinese academy of agricultural sciences

<120> application of dye decolorization oxidase StDyP for simultaneously degrading aflatoxin and zearalenone

<160> 4

<170> SIPOSequenceListing 1.0

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<213> Streptomyces thermocarboxydus

<400> 1

Met Ala Asp Pro Ser Leu Ser Gln Thr Arg Thr Pro Glu Lys Glu Pro

1 5 10 15

Gln Ala Glu Ala Ala Ala Ser Gly Ile Ser Arg Arg Arg Leu Leu Gly

20 25 30

Thr Ala Gly Ala Thr Gly Leu Val Leu Gly Ala Ala Gly Gly Ala Val

35 40 45

Gly Tyr Ala Ser Ala Pro Thr Gly Ala Thr Pro Leu Thr Ser Val Gly

50 55 60

Ala Thr Lys Val Pro Phe His Val Lys His Gln Pro Gly Ile Thr Asp

65 70 75 80

Pro Leu Gln Ser Arg Gly His Leu Leu Ala Phe Asp Leu Arg Pro Gly

85 90 95

Ala Gly Arg Lys Glu Ala Ala Ala Leu Leu Arg Arg Trp Ser Asp Thr

100 105 110

Ala Arg Arg Leu Met Asp Gly Thr Phe Asp Ala Glu Gly Asp Ser Asp

115 120 125

Val Ala Arg Asp Ala Gly Pro Ser Ser Leu Thr Leu Thr Phe Gly Phe

130 135 140

Gly His Ser Phe Phe Ala Arg Thr Gly Leu Glu Arg Gln Arg Pro Ala

145 150 155 160

Ala Leu Glu Pro Leu Pro Ala Phe Ser Ser Asp Arg Leu Asp Arg Ala

165 170 175

Arg Ser Asp Gly Asp Leu Trp Val Gln Ile Gly Ala Asp Asp Ala Leu

180 185 190

Val Ala Phe His Ala Leu Arg Ala Val Gln Lys Asp Ala Gly Ala Ala

195 200 205

Ala Arg Val Arg Trp Gln Met Asn Gly Phe Asn Arg Ser Pro Gly Ala

210 215 220

Thr Ala Arg Pro Met Thr Thr Arg Asn Leu Met Gly Gln Val Asp Gly

225 230 235 240

Thr Arg Asn Pro Lys Pro Asp Glu Pro Asp Phe Asp Gln Arg Ile Phe

245 250 255

Val Ala Glu Gln Gly Glu Pro Ala Trp Met Ala Asn Gly Ser Tyr Val

260 265 270

Val Val Arg Arg Ile Arg Met Leu Leu Asp Asp Trp Glu Lys Leu Ser

275 280 285

Leu Arg Glu Gln Glu Gly Val Ile Gly Arg Arg Lys Ala Asp Gly Ala

290 295 300

Pro Leu Ser Gly Gly Asp Glu Thr Thr Glu Met Asp Leu Glu Lys Thr

305 310 315 320

Asp Ala Gln Gly Asn Leu Val Val Pro Phe Asn Ala His Ala Arg Ile

325 330 335

Thr Arg Pro Asp Gln Asn Gly Gly Ala Ala Met Leu Arg Arg Pro Phe

340 345 350

Ser Tyr His Asp Gly Ile Asp Ala Asp Gly Thr Pro Asp Ala Gly Leu

355 360 365

Leu Phe Ile Cys Trp Gln Ala Asp Pro Leu Arg Gly Phe Val Pro Val

370 375 380

Gln Arg Lys Leu Asp Arg Gly Asp Ala Leu Thr Pro Phe Ile Arg His

385 390 395 400

Glu Ala Ser Gly Leu Phe Ala Val Pro Gly Gly Ala Ala Glu Gly Glu

405 410 415

Tyr Val Gly Gln Ala Leu Leu Glu Gly

420 425

<210> 2

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<213> Streptomyces thermocarboxydus

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gccgcctccg gcatctcgcg gcggcgcctg ctcggcacgg ccggcgccac cgggctcgtg 120

ctcggggcgg ccggcggtgc cgtcgggtac gcgtcggcgc ccaccggagc cactccgctc 180

acctcggtcg gcgccacaaa agtcccgttt cacgtgaaac atcagccggg catcaccgac 240

ccgctccagt cgcgtggcca tctcctcgcc ttcgacctga ggcccggcgc cggacgcaag 300

gaggcggctg cgctgctgcg ccgctggtcc gacaccgccc ggcggctgat ggacgggacg 360

ttcgacgccg agggcgacag tgacgtggcc cgtgacgcgg ggccctcctc gctgaccctg 420

accttcggtt ttgggcacag cttcttcgcg cgcaccgggc tggagaggca gcgtccggcc 480

gccctggagc cgctgcccgc cttctcctcc gaccgcctcg accgggcccg cagcgacggg 540

gacctgtggg tgcagattgg cgccgacgac gccctcgtcg cgttccatgc cctgcgcgcg 600

gtgcagaagg acgcgggcgc ggcggcccgg gtgcgctggc agatgaacgg cttcaaccgg 660

tcgccgggcg ccaccgcccg cccgatgacc acccgcaatc tgatgggcca ggtcgacggc 720

acccgcaacc cgaaacccga cgagcccgac ttcgaccagc ggatcttcgt ggcggagcag 780

ggcgagcccg cctggatggc gaacggctcc tatgtggtcg tccgccggat ccgcatgctg 840

ctggacgact gggagaagct gtcgctcagg gagcaggagg gtgtcatcgg gcggcgcaag 900

gcggacggcg ccccgctctc cgggggcgac gagacgaccg agatggacct ggagaagacc 960

gacgcccagg gcaatctggt cgtcccgttc aacgcgcacg cacgcatcac ccggcccgac 1020

cagaacggcg gggcggcgat gctgcgccgg ccgttctcgt accacgacgg catcgacgcg 1080

gacgggacgc cggacgcggg gctgctgttc atctgctggc aggccgatcc gctgcgcggc 1140

ttcgtgccgg tgcagcgcaa gctcgaccgg ggcgacgccc tgacgccgtt catccgtcac 1200

gaggcgagcg ggctgttcgc cgtcccgggc ggggcggcgg agggtgagta tgtgggccag 1260

gcgctgctgg aggggtga 1278

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<212> DNA

<213> Artificial Sequence (Artificial Sequence)

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<212> DNA

<213> Artificial Sequence (Artificial Sequence)

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ttttaagcag agattaccta tctagacccc tccagcagcg cctggccc 48