CN111073867B

CN111073867B - Dye decolorization peroxidase BsDyP and application thereof in mycotoxin detoxification

Info

Publication number: CN111073867B
Application number: CN202010013737.2A
Authority: CN
Inventors: 赵丽红; 郭永鹏; 马秋刚; 计成; 唐彧
Original assignee: China Agricultural University
Current assignee: China Agricultural University
Priority date: 2020-01-07
Filing date: 2020-01-07
Publication date: 2022-04-08
Anticipated expiration: 2040-01-07
Also published as: CN111073867A

Abstract

The invention belongs to the technical field of agricultural biology, and particularly relates to dye decoloration peroxidase BsDyP, and a coding gene and application thereof; the amino acid sequence of the dye decolorizing peroxidase BsDyP is shown as SEQ ID NO.1 or SEQ ID NO. 2; the invention also discloses a gene for coding the BsDyP, and the DNA sequence of the gene is shown as SEQ ID NO.3 or SEQ ID NO. 4; the invention also discloses a preparation method of the dye decolorizing peroxidase BsDyP and application of the dye decolorizing peroxidase BsDyP in mycotoxin detoxification, and a preparation method and application of the dye decolorizing peroxidase BsDyP as a mycotoxin biodegradation agent.

Description

Dye decolorization peroxidase BsDyP and application thereof in mycotoxin detoxification

Technical Field

The invention belongs to the technical field of agricultural biology, and particularly relates to dye decolorization peroxidase BsDyP, and a coding gene and application thereof, in particular to application in mycotoxin detoxification.

Background

Mycotoxins are secondary metabolites of fungi, have carcinogenic, genotoxic, reproductive, neurotoxic and immunosuppressive effects, and are extremely harmful to humans and various livestock and poultry. There are over 300 mycotoxins that have been isolated and identified to date, with the most widespread contamination and toxicity, and the most studied being Aflatoxins (AFT), Zearalenone (ZEN), trichothecenes (trichothecenes, including vomitoxin and T-2 toxin, among others), ochratoxins (ochratoxins) and fumonisins (fumonisins), among others. At present, the methods for detoxifying mycotoxins in the food and feed industry mainly comprise two main types, one is physical adsorption detoxification, and some inorganic adsorbents of montmorillonite and organic adsorbents of esterified mannan are added; the other is biodegradation, which is to biodegrade mycotoxin by microorganisms or enzymes produced by the microorganisms, destroy the molecular structure of the toxin and metabolize the toxin to generate non-toxic or low-toxic metabolites. The biodegradation method is safe, efficient and environment-friendly, and is a hot point for researching the detoxification of mycotoxins.

The enzymes reported to have the activity of degrading zearalenone currently include lactonohydrolase ZHD101 derived from Gliocladium roseum and zearalenone degrading enzyme ZENdease-N2 derived from Rhizoctonia solani; the reported enzymes for degrading aflatoxin comprise aflatoxin oxidase ADTZ derived from Armillaria pseudomeliloti, F420H2 dependent reductase FDR derived from mycobacteria and fungal laccase derived from white rot fungi, but the degradation mechanism of the enzymes and the toxicity of degradation products are not clear, so that the research and application of the fungal toxin degrading enzyme are limited. There is increasing evidence that other enzymes with unknown properties may also be involved in the biodegradation of mycotoxins, both zearalenone and aflatoxin, which have benzene and lactone ring structures. Therefore, finding and exploring new mycotoxin degrading enzymes is of great practical significance.

Disclosure of Invention

In view of the above, the present invention provides a dye decolorizing peroxidase BsDyP with a function of degrading mycotoxin and an application thereof in mycotoxin detoxification. The dye decolorizing peroxidase BsDyP can degrade mycotoxin.

The invention aims to provide dye decolorizing peroxidase BsDyP with a function of degrading mycotoxin.

Another object of the present invention is to provide the above-mentioned gene encoding dye decolorizing peroxidase BsDyP having a function of degrading mycotoxins.

Another object of the present invention is to provide a recombinant expression vector containing the above-mentioned coding gene.

Another object of the present invention is to provide a recombinant strain or cell containing the above-mentioned encoding gene.

The invention also aims to provide a preparation method of the dye decolorizing peroxidase BsDyP.

The invention also aims to provide application of the dye decolorizing peroxidase BsDyP with the function of degrading mycotoxin.

The invention also aims to provide a compound enzyme containing the dye decolorizing peroxidase BsDyP and the glucose oxidase GOD.

The invention also aims to provide application of the complex enzyme containing the dye decolorizing peroxidase BsDyP and glucose oxidase.

Another object of the present invention is to provide a mycotoxin biodegradation agent containing the dye decolorizing peroxidase BsDyP.

The invention also aims to provide a preparation method of the mycotoxin biodegradation agent containing dye decolorizing peroxidase BsDyP.

It is another object of the present invention to provide a method for degrading mycotoxins.

The invention also aims to provide a compound enzyme containing the dye decolorizing peroxidase BsDyP, the dye decolorizing peroxidase BsDyP and the glucose oxidase GOD, and application of a mycotoxin biodegradation agent in mycotoxin detoxification.

In order to achieve the above object, the present invention provides the following technical solutions:

the amino acid sequence of the dye decolorizing peroxidase BsDyP with the function of degrading the mycotoxin can be as follows:

a sequence shown as SEQ ID NO.1 or SEQ ID NO.2, wherein SEQ ID NO.1 contains a signal peptide and SEQ ID NO.2 does not contain a signal peptide;

or one or two amino acid residues in the sequence shown in SEQ ID NO.1 or SEQ ID NO.2 are substituted and/or deleted and/or inserted;

or a sequence which has at least 90% of sequence identity, preferably 95% of sequence identity, with the amino acid sequence shown in SEQ ID NO.1 or SEQ ID NO.2 and has the function of dye decolorizing peroxidase BsDyP.

The invention also provides a nucleotide sequence for coding the dye decolorizing peroxidase BsDyP, which is as follows:

a sequence shown as SEQ ID NO.3 or SEQ ID NO. 4;

or one or two nucleotide residues in the sequence shown in SEQ ID NO.3 or SEQ ID NO.4 are substituted and/or deleted and/or inserted;

or a nucleotide sequence having at least 90% sequence identity, preferably 95% sequence identity, with the nucleotide sequence shown in SEQ ID No.3 or SEQ ID No. 4.

SEQ ID NO.1：

MSDEQKKPEQIHRRDILKWGAMAGAAVAIGASGLGGLAPLVQTAAKPSKKDEKEEEQIVPFYGKHQAGITTAHQTYVYFAALDVTAKDKSDIITLFRNWTSLTQMLTSGKKMSAEQRNQYLPPQDTGESADLSPSNLTVTFGFGPGFFEKDGKDRFGLKSKKPKHLAALPAMPNDNLDEKQGGGDICIQVCADDEQVAFHALRNLLNQAVGTCEVRFVNKGFLSGGKNGETPRNLFGFKDGTGNQSTKDDTLMNSIVWIQSGEPDWMTGGTYMAFRKIKMFLEVWDRSSLKDQEDTFGRRKSSGAPFGQKKETDPVKLNQIPSNSHVSLAKSTGKQILRRAFSYTEGLDPKTGYMDAGLLFISFQKNPDNQFIPMLKALSAKDALNEYTQTIGSALYACPGGCKKGEYIAQRLLES

SEQ ID NO.2：

IGASGLGGLAPLVQTAAKPSKKDEKEEEQIVPFYGKHQAGITTAHQTYVYFAALDVTAKDKSDIITLFRNWTSLTQMLTSGKKMSAEQRNQYLPPQDTGESADLSPSNLTVTFGFGPGFFEKDGKDRFGLKSKKPKHLAALPAMPNDNLDEKQGGGDICIQVCADDEQVAFHALRNLLNQAVGTCEVRFVNKGFLSGGKNGETPRNLFGFKDGTGNQSTKDDTLMNSIVWIQSGEPDWMTGGTYMAFRKIKMFLEVWDRSSLKDQEDTFGRRKSSGAPFGQKKETDPVKLNQIPSNSHVSLAKSTGKQILRRAFSYTEGLDPKTGYMDAGLLFISFQKNPDNQFIPMLKALSAKDALNEYTQTIGSALYACPGGCKKGEYIAQRLLES

SEQ ID NO.3：

ATGAGCGATGAACAGAAAAAGCCAGAACAAATTCACAGACGGGACATTTTAAAATGGGGAGCGATGGCGGGGGCAGCCGTTGCGATCGGTGCCAGCGGTCTCGGCGGTCTCGCTCCGCTTGTTCAGACTGCGGCTAAGCCATCGAAAAAGGATGAAAAAGAGGAGGAGCAGATTGTTCCGTTTTACGGAAAGCATCAGGCCGGAATCACAACTGCCCATCAGACGTATGTCTATTTTGCTGCACTGGATGTAACAGCAAAAGACAAAAGCGACATCATTACGTTATTTAGAAACTGGACAAGTCTCACACAGATGCTGACGTCTGGCAAGAAAATGTCAGCGGAGCAGAGAAATCAGTATCTGCCGCCGCAGGATACAGGTGAGTCTGCTGATTTATCCCCTTCCAACTTAACGGTCACATTCGGATTCGGGCCTGGCTTTTTTGAAAAAGACGGAAAGGACCGCTTCGGACTGAAAAGCAAAAAACCGAAGCACCTAGCCGCTCTTCCAGCGATGCCCAATGACAACCTGGATGAAAAGCAGGGAGGCGGGGACATCTGCATTCAAGTGTGTGCAGACGACGAGCAAGTGGCGTTTCACGCGCTGCGTAACCTGCTAAATCAAGCGGTCGGCACTTGTGAGGTCCGTTTTGTGAACAAAGGCTTTTTAAGCGGTGGAAAAAATGGTGAAACACCGCGCAACCTCTTCGGGTTTAAAGATGGAACAGGCAACCAGAGCACGAAGGATGACACGTTGATGAACTCGATCGTGTGGATTCAGTCTGGTGAGCCTGACTGGATGACGGGCGGCACCTATATGGCTTTTCGGAAAATCAAAATGTTCCTTGAGGTATGGGACCGCTCTTCCCTCAAGGACCAAGAGGATACCTTCGGCCGCAGAAAAAGCTCGGGGGCGCCTTTTGGGCAGAAAAAGGAAACAGATCCCGTGAAGCTGAATCAAATTCCGTCTAATTCACACGTCAGTCTAGCGAAATCTACTGGGAAACAAATTTTGCGAAGAGCTTTCTCTTACACAGAAGGACTTGATCCGAAAACCGGCTATATGGATGCAGGTCTCCTGTTTATCAGTTTTCAAAAAAATCCCGACAATCAGTTTATCCCGATGCTGAAGGCTCTTTCAGCAAAGGATGCGTTAAACGAATATACACAAACAATCGGTTCTGCTTTATACGCATGCCCAGGCGGCTGCAAAAAAGGAGAATATATTGCCCAGCGTTTGCTGGAATCATAA

SEQ ID NO.4：

ATCGGTGCCAGCGGTCTCGGCGGTCTCGCTCCGCTTGTTCAGACTGCGGCTAAGCCATCGAAAAAGGATGAAAAAGAGGAGGAGCAGATTGTTCCGTTTTACGGAAAGCATCAGGCCGGAATCACAACTGCCCATCAGACGTATGTCTATTTTGCTGCACTGGATGTAACAGCAAAAGACAAAAGCGACATCATTACGTTATTTAGAAACTGGACAAGTCTCACACAGATGCTGACGTCTGGCAAGAAAATGTCAGCGGAGCAGAGAAATCAGTATCTGCCGCCGCAGGATACAGGTGAGTCTGCTGATTTATCCCCTTCCAACTTAACGGTCACATTCGGATTCGGGCCTGGCTTTTTTGAAAAAGACGGAAAGGACCGCTTCGGACTGAAAAGCAAAAAACCGAAGCACCTAGCCGCTCTTCCAGCGATGCCCAATGACAACCTGGATGAAAAGCAGGGAGGCGGGGACATCTGCATTCAAGTGTGTGCAGACGACGAGCAAGTGGCGTTTCACGCGCTGCGTAACCTGCTAAATCAAGCGGTCGGCACTTGTGAGGTCCGTTTTGTGAACAAAGGCTTTTTAAGCGGTGGAAAAAATGGTGAAACACCGCGCAACCTCTTCGGGTTTAAAGATGGAACAGGCAACCAGAGCACGAAGGATGACACGTTGATGAACTCGATCGTGTGGATTCAGTCTGGTGAGCCTGACTGGATGACGGGCGGCACCTATATGGCTTTTCGGAAAATCAAAATGTTCCTTGAGGTATGGGACCGCTCTTCCCTCAAGGACCAAGAGGATACCTTCGGCCGCAGAAAAAGCTCGGGGGCGCCTTTTGGGCAGAAAAAGGAAACAGATCCCGTGAAGCTGAATCAAATTCCGTCTAATTCACACGTCAGTCTAGCGAAATCTACTGGGAAACAAATTTTGCGAAGAGCTTTCTCTTACACAGAAGGACTTGATCCGAAAACCGGCTATATGGATGCAGGTCTCCTGTTTATCAGTTTTCAAAAAAATCCCGACAATCAGTTTATCCCGATGCTGAAGGCTCTTTCAGCAAAGGATGCGTTAAACGAATATACACAAACAATCGGTTCTGCTTTATACGCATGCCCAGGCGGCTGCAAAAAAGGAGAATATATTGCCCAGCGTTTGCTGGAATCATAA

The invention also provides a recombinant vector containing the dye decolorizing peroxidase BsDyP, preferably PET-31 b-BsDyP.

The invention also provides a recombinant strain containing the dye decolorizing peroxidase BsDyP, preferably the recombinant strain Rosseta (DE 3)/BsDyP.

The invention also provides a method for preparing the dye decolorizing peroxidase BsDyP, which comprises the following steps:

(1) transforming host cells by using a recombinant expression vector containing a gene coding dye decolorizing peroxidase BsDyP to obtain a recombinant strain;

(2) culturing the recombinant strain, and inducing dye decolorizing peroxidase BsDyP to express;

(3) and obtaining the purified dye decolorizing peroxidase BsDyP by utilizing nickel ion affinity chromatography.

The invention also provides application of the dye decolorizing peroxidase BsDyP, particularly application in the aspect of toxin detoxification of mycotoxin, wherein the mycotoxin includes but is not limited to zearalenone, aflatoxin, trichothecene toxoid (including vomitoxin and T-2 toxin), ochratoxin, fumonisin, patulin, gliotoxin and variegated aspergillin, and the mycotoxin can be directly degraded or degraded depending on a mediator, such as zearalenone, zearalenol, zearalanol, aflatoxin B1, B2, G1, G2, M1 or M2.

The invention also provides a complex enzyme, which comprises dye decolorizing peroxidase BsDyP and glucose oxidase GOD.

The invention also provides application of the complex enzyme containing dye decolorizing peroxidase BsDyP and glucose oxidase GOD, in particular application in mycotoxin detoxification, which can directly degrade or degrade zearalenone, zearalenol, zearalanol, aflatoxin B1, B2, G1, G2, M1 or M2 and other mycotoxins depending on a mediator.

The invention also provides a mycotoxin biodegradation agent, which comprises dye decolorizing peroxidase BsDyP and a physiologically acceptable compatible carrier and/or a mediator, wherein the physiologically acceptable compatible carrier comprises but is not limited to maltodextrin, cyclodextrin, wheat bran, rice bran, sucrose, starch and Na₂SO₄One or more of talc, montmorillonite, oligosaccharide or yeast cell wall; the mediator is selected from one or more of syringaldehyde, acetosyringone, acetovanillone, p-coumaric acid, methyl syringate, carvacrol, thymol, cinnamaldehyde, citral, limonene, gamma-terpinene, perillaldehyde, allicin, eugenol, anethole, menthone, linalool, isovaleraldehyde, linalool, zingerone, isothiocyanate, 2-hydrazine-bis (3-ethyl-benzothiazole-6-sulfonic acid) ABTS, violuric acid VIO, 1-hydroxybenzotriazole HBT, 2 ', 6, 6' -tetramethylpiperidine oxide TEMPO or polyoxometallate.

The mycotoxin biodegradation agent also comprises a microecological preparation, wherein the microecological preparation comprises but is not limited to one or more of enterococcus faecalis, enterococcus faecium, bacillus licheniformis, bacillus subtilis, lactobacillus plantarum, pediococcus acidilactici, bifidobacterium bifidum, enterococcus acidilactici, lactobacillus acidophilus, lactobacillus casei, lactobacillus delbrueckii subsp lactis, pediococcus pentosaceus, candida utilis, bacillus lentus, bacillus pumilus, bacillus amyloliquefaciens, bifidobacterium longum, bifidobacterium breve, bifidobacterium adolescentis, streptococcus thermophilus, lactobacillus reuteri, bifidobacterium animalis, aspergillus oryzae, lactobacillus cellobiosus, lactobacillus fermentum or lactobacillus delbrueckii subsp bulgaricus, preferably, the mycotoxin biodegradation agent also comprises lactobacillus buchneri, lactobacillus bulgaricus, or lactobacillus delbrueckii when used for silage or cattle feed, At least one of propionibacterium and lactobacillus paracasei; or the mycotoxin biodegradation agent also contains bacillus coagulans and/or brevibacillus laterosporus when being used for feeds of poultry, pigs and aquaculture animals.

The mycotoxin biodegrading agent may further comprise additional enzymes including, but not limited to, zearalenone lactonase, fumonisin carboxyl esterase, fumonisin aminotransferase, aminopolyolamine oxidase, aflatoxin oxidase, laccase, carboxypeptidase, aspergillus niger aspartic protease, elastase, aminopeptidase, pepsin-like protease, trypsin-like protease, bacterial protease, amylase, arabinase, arabinofuranosidase, cellulase, hemi-cellulase, lignin-degrading enzyme, chitinase, rennin, cutinase, deoxyribonuclease, epimerase, esterase, galactosidase, glucanase, endoglucanase, glucoamylase, glucosidase, glucuronidase, hexose oxidase, elastase, aminopeptidase, and pepsin, One or more of superoxide dismutase, hydrolase, transferase, isomerase, lipolytic enzyme, lyase, mannosidase, xylanase, glutathione peroxidase, oxidoreductase, aldone reductase, pectin acetyl esterase, pectin depolymerase, pectin methylesterase, pectinolytic enzyme, manganese peroxidase, phenol oxidase, phytase, polygalacturonase, alcohol dehydrogenase, ribonuclease, transglutaminase, epoxide hydrolase, and acid phosphatase.

The invention also provides a method for degrading mycotoxin, which comprises the step of treating a material containing the mycotoxin by using the dye decolorizing peroxidase BsDyP or the compound enzyme or the mycotoxin biodegradation agent.

In the above method, the treatment is mixing the above dye decolorizing peroxidase BsDyP or the above complex enzyme or the above mycotoxin biodegradation agent with a material containing mycotoxin.

In the above method, the mycotoxins include but are not limited to zearalenone, aflatoxins, trichothecene toxoids (including vomitoxin, T-2 toxin), ochratoxins, fumonisins, patulin, gliotoxins, and variegated aspergillons, especially zearalenone, zearalenol, zearalanol, aflatoxins B1, B2, G1, G2, M1, or M2.

In the above method, the material containing mycotoxin includes, but is not limited to, food, feed and raw materials, grains and processing byproducts, grain oil, milk, aged grains, tea leaves, fruits and fruit juice, Chinese herbal medicines, etc., and includes various materials containing mycotoxin.

The invention has the advantages and beneficial effects that:

the invention provides dye decolorizing peroxidase BsDyP with a function of degrading mycotoxin, and a coding gene and application thereof, and particularly relates to application thereof in the aspect of degrading mycotoxin. The dye decolorizing peroxidase BsDyP is reported for the first time to be capable of degrading mycotoxin, and the enzyme has high catalysis efficiency and high conversion efficiency, is derived from probiotic bacillus and has very wide application prospect in feed and food industries.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.

FIG. 1 is a SDS-PAGE picture of the purified expression product of recombinant plasmid PET31 b-BsDyP; wherein lane 1 is purified recombinant dye decolorized peroxidase BsDyP, lane M is protein molecular weight standards (116, 66.2, 45, 35, 25, 18.4, 14.4 kDa);

FIG. 2 shows the degradation of ZEN by recombinant dye decolorizing peroxidase rBsDyP under different temperature conditions;

FIG. 3 shows the degradation of ZEN by recombinant dye decolorizing peroxidase rBsDyP under different pH conditions;

FIG. 4 shows the degradation of ZEN by recombinant dye decolorizing peroxidase rBsDyP under the participation of mediator ABTS;

FIG. 5 shows the degradation of AFB1 by recombinant dye decolorizing peroxidase rBsDyP under conditions in which the mediator ABTS participates;

FIG. 6 shows the degradation of ZEN by recombinant dye decolorizing peroxidase rBsDyP in combination with different concentrations of glucose oxidase.

Detailed Description

The invention discloses dye decolorizing peroxidase BsDyP with a function of degrading mycotoxin, a coding gene thereof and application thereof in mycotoxin detoxification. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.

The main experimental materials and reagents used in the examples of the invention were:

coli expression vector PET-31b, the cloned strain E.coli DH5 a, and the expression strain E.coli Rosseta (DE3) were purchased from Invitrogen. Restriction endonucleases and DNA ligases were purchased from NEB, zearalenone and aflatoxin standards from sigma, and other reagents were home-made analytical grade.

The biochemical reagents used in the examples are all commercially available reagents, and the technical means used in the examples are conventional means used by those skilled in the art, unless otherwise specified.

The invention is further illustrated by the following examples:

example 1 dye decolorizing peroxidase BsDyP acquisition and expression

The method comprises the steps of taking Bacillus subtilis 168 genome DNA as an amplification template, amplifying a coding gene of BsDyP, constructing a recombinant expression vector containing a BsDyP coding gene sequence and engineering bacteria thereof, and expressing BsDyP. The method comprises the following specific steps:

1. cloning of BsDyP gene encoding dye decolorizing peroxidase

1.1 extraction of Bacillus subtilis genomic DNA

(1) The resulting mixture was inoculated from a glycerol tube and streaked onto an LB solid plate, and then subjected to static culture at 37 ℃ for 12 hours.

(2) A single colony was picked from the plate on which the cells were cultured and inoculated into 5mL of liquid LB medium and cultured at 37 ℃ at 180r/min for 12 hours.

(3) The bacterial liquid is subpackaged into a sterilized 1.5mL microcentrifuge tube, centrifuged at 12000r/min for 1min to collect the thallus, and the supernatant is discarded.

(4) Adding 500 μ L of cell suspension into the centrifuge tube with thallus precipitate, blowing with a gun head to suspend thallus, and bathing at 37 deg.C for 60 min; the cells were collected by centrifugation at 12000r/min for 1min and the supernatant was discarded.

(5) 225. mu.L of buffer A was added to the pellet, and the pellet was shaken until the pellet was completely suspended.

(6) Add 10. mu.L proteinase K solution to the tube and mix by inversion.

(7) Adding 25 mu L of lysis solution S, reversing and uniformly mixing; standing in water bath at 57 deg.C for 20min, and mixing by reversing for 1-2 times.

(8) Add 250. mu.L of buffer B and mix well with shaking for 5 s.

(9) Add 250. mu.L of absolute ethanol, shake well and mix for 15 s.

(10) Adding the solution and flocculent precipitate obtained in the previous step into an adsorption column, centrifuging at 12000r/min for 30s, pouring off waste liquid, and placing the adsorption column into a collecting pipe.

(11) Adding 500 μ L buffer solution C into adsorption column, centrifuging at 12000r/min for 30s, pouring off waste liquid, and placing adsorption column into collection tube.

(12) Add 700. mu.L of buffer W2 to the adsorption column, centrifuge for 30s, pour off the waste, place the adsorption column in the collection tube.

(13) The adsorption column was added with 500. mu.L of buffer W2, centrifuged at 12000r/min for 3min, and the waste solution was discarded.

(14) Placing the adsorption column in a clean centrifuge tube, standing at room temperature for several minutes, suspending and dropwise adding 150 μ L of eluent TE to the middle part of the adsorption membrane, standing at room temperature for 5min, centrifuging at 12000r/min for 2min, and collecting the solution in the centrifuge tube.

1.2 the Bacillus subtilis dye decoloration peroxidase BsDyP coding gene is amplified, and the steps are as follows:

an upstream primer P1 and a downstream primer P2 were designed according to the multi-cloning site of the vector PET-31b by selecting NdeI and XhoI as the restriction sites, and were synthesized by Shanghai Biotechnology Ltd. The sequences of the upstream primer P1 and the downstream primer P2 are designed as follows:

upstream primer P1: 5'-GCTTATTCATATGATCGGTGCCA-3' (shown as SEQ ID NO. 5)

The downstream primer P2: 5'-GTTCTCGAGTGATTCCAGCA-3' (shown as SEQ ID NO. 6)

The Bacillus subtilis genome DNA is used as a template for amplification, and the reaction conditions are as follows:

DNA template	1μL
		Upstream primer P1	2μL
Downstream primer P2	2μL
		2×Pfu PCR Mix	25μL
ddH₂O₂	20μL
		Total volume	50μL

The amplification conditions were: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30 s; annealing at 50 ℃ for 30s, and extending at 72 ℃ for 1min for 30s to react for 30 cycles; extension was complete for 10min at 72 ℃. The PCR amplification product was subjected to electrophoresis on a 1% agarose gel, and the PCR product was recovered using an agarose gel DNA recovery kit.

2. The recombinant expression vector containing dye decolorizing peroxidase BsDyP coding gene sequence is constructed by using NdeI and XhoI to double-enzyme-cut PCR products recovered in the previous step and PET-31b plasmid, and the enzyme cutting system is as follows:

PCR product/PET-31 b plasmid	30μL
		NdeI	1μL
XhoI	1μL

10×CutSmart Buffer	5μL
		ddH₂O₂	13μL
Total volume	50μL

The enzyme digestion conditions are as follows: water bath at 37 deg.c for 30 min. The digested product was electrophoresed through 1% agarose gel, and the PCR product and the plasmid digested fragment were recovered by agarose gel DNA recovery kit.

Connecting the PCR product and the plasmid recovered by enzyme digestion with T4DNA ligase at 16 ℃ overnight; and transforming the connecting product into an escherichia coli competent cell DH5 alpha, screening by an Amp resistance plate, selecting a positive transformant, extracting a transformation plasmid, carrying out single-enzyme digestion and double-enzyme digestion verification and sequencing, and determining to construct and obtain a correct recombinant strain DH5 alpha/PET-31 b-BsDyP.

3. Induced expression and purification of recombinant dye decolorization peroxidase rBsDyP in escherichia coli

3.1 recombinant dye decolorization peroxidase rBsDyP induced expression

Recombinant E.coli Rosseta (DE3) transformed with PET-31b-BsDyP plasmid was inoculated in 5mL of liquid LB medium and activated overnight at a rate of 1: transferring 100 proportion into 500mL triangular flask with liquid loading capacity of 300mL, culturing at 37 ℃ at 180r/min until OD600 is 0.6, and adding IPTG with final concentration of 0.2mM to induce target protein expression at 28 ℃.

3.2 recombinant dye decolorization peroxidase rBsDyP purification

Collecting fermentation liquor, centrifuging at 4 deg.C and 12000rpm for 30min, and removing supernatant; the cells were resuspended in phosphate buffer pH7.0, centrifuged at 12000rpm for 30min at 4 ℃ and the supernatant discarded, and the cells were washed three times. Then, the bacterial cells were resuspended in a binding buffer of 1mg/mL, disrupted by sonication, centrifuged at 12000rpm for 10min at 4 ℃ and the supernatant was collected and filtered. Since the expressed rBsDyP protein was tagged with histidine at the C-terminus (6 XHis), a nickel ion affinity chromatography column (Ni 2) was used⁺NTA) purification of recombinant proteins, equilibration, loading, elution, etc., see Qiagen instructions. The purified protein was ultrafiltered with a retention tube (3kDa) to remove imidazole contained therein, and the purification result of the target protein was detected by SDS-PAGE electrophoresis, and the result is shown in FIG. 1, wherein lane 1 is the target band of the expression product, and the molecular weight of the protein is about 44kDa, which is consistent with the theoretical molecular weight.

Example 2 recombinant dye decolorizing peroxidase rBsDyP degradation of zearalenone under different temperature conditions

Dissolving Zearalenone (ZEN) in methanol to obtain 1000 μ g/mL stock solution, and slowly adding 1mL sodium phosphateThe degradation reaction was carried out in a wash (0.1M, pH8.0) system, the rBsDyP protein concentration obtained in example 1 was 100. mu.g/mL, the ZEN concentration was 10. mu.g/mL, and H in the reaction system₂O₂The content was 100. mu.M, and the system without addition of rBsDyP protein was used as a control; respectively reacting at 27, 32, 37, 42, 47, 52 and 57 ℃ for 1h, adding 1mL of methanol to terminate the reaction, and detecting the ZEN content by adopting high performance liquid chromatography.

The chromatographic conditions for detecting ZEN by high performance liquid chromatography are as follows: a chromatographic column: agilent C18 chromatography column, 4.6mm × 150mm × 5 μm; mobile phase: acetonitrile-water-methanol (46: 46: 8); flow rate: 1 mL/min; pumping pressure: 45 bar; sample introduction amount: 20 mu L of the solution; fluorescence detector detection wavelength: λ ex-274 nm, λ em-440 nm; collecting time: for 18 minutes.

As shown in FIG. 2, the optimum temperature for degradation of ZEN by rBsDyP protein was 42 ℃ and the degradation rate of rBsDyP protein obtained in example 1 was 83% after reacting with ZEN (10. mu.g/mL) for 1 hour under the optimum temperature condition.

Example 3 decolorization of recombinant dyes peroxide rBsDyP degradation of zearalenone at different pH conditions

To test the activity of rBsDyP to degrade ZEN prepared in example 1 under different pH conditions, degradation was performed in 1mL of buffers (pH4-6, 0.1M sodium citrate buffer; pH7-8, 0.1M sodium phosphate buffer; pH9-10, 0.1M glycine-NaOH buffer) at different pH values, in which H was added₂O₂The content was 100. mu.M, the rBsDyP protein concentration obtained in example 1 was 100. mu.g/mL, and the ZEN concentration was 10. mu.g/mL; a system without addition of the rBsDyP protein was used as a control; reacting at 42 ℃ for 1h, adding 1mL of methanol to terminate the reaction, and detecting the ZEN content by adopting high performance liquid chromatography.

As shown in FIG. 3, rBsDyP protein degraded ZEN with the optimum pH of 8, and rBsDyP prepared in example 1 was reacted with ZEN (10. mu.g/mL) at the optimum pH for 1 hour to achieve a degradation rate of 83%.

Example 4 degradation of ZEN by recombinant dye decolorizing peroxidase rBsDyP in the Presence of the mediator ABTS

In 1mL of buffer solution with different pH values (pH4-6, 0.1M sodium citrate buffer solution; pH7-8, 0.1M sodium phosphate buffer solution; pH9-10, 0.1M glycineacid-NaOH buffer solution) and H in the reaction system₂O₂The content was 100. mu.M, the rBsDyP protein concentration prepared in example 1 was 100. mu.g/mL, the ZEN concentration was 10. mu.g/mL, and the ABTS content was 1mM or 5 mM; a system without addition of the rBsDyP protein was used as a control; after reacting for 1h at 42 ℃, 1mL of methanol is added to stop the reaction, and the ZEN content is detected by adopting high performance liquid chromatography.

As shown in FIG. 4, the degradation of ZEN by rBsDyP can be enhanced by adding ABTS, and when ABTS exists in the system, the degradation rate of ZEN is more than 95% under the conditions of pH5 to 10 after the reaction is carried out for 1 h. Under the condition of pH4, when the ABTS content in the system is 1mM, the ZEN degradation rate is 52%; when the ABTS content in the system is 5mM, the ZEN degradation rate is 85%.

Example 5 degradation of AFB1 by recombinant dye decolorizing peroxidase rBsDyP in the Presence of the mediator ABTS

Performing degradation reaction in 1mL buffer solution (pH4-6, 0.1M sodium citrate buffer solution; pH7-8, 0.1M sodium phosphate buffer solution; pH9-10, 0.1M glycine-NaOH buffer solution) with different pH values, wherein H in the reaction system₂O₂The content is 100 mu M, the concentration of the rBsDyP protein prepared in example 1 is 100 mu g/mL, the concentration of AFB1 is 2 mu g/mL, and the content of ABTS is 1mM or 5 mM; a system without addition of the rBsDyP protein was used as a control; after reacting for 1h at 42 ℃, 1mL of methanol is added to stop the reaction, and the content of AFB1 is detected by high performance liquid chromatography.

The results are shown in FIG. 5, and under neutral and alkaline conditions (pH 7 to 10), when the ABTS content in the system is 1mM, the degradation rate of AFB1 exceeds 74%; when the ABTS content in the system is 5mM, the degradation rate of AFB1 exceeds 85 percent.

Example 6 recombinant dye decolorizing peroxidase rBsDyP in combination with varying concentrations of glucose oxidase GOD for zearalenone degradation

To test the effect of rBsDyP prepared in example 1 and glucose oxidase GOD with different concentrations in combination to degrade ZEN, 1mL of the sodium phosphate buffer (0.1M, pH8.0) reaction system used in example 2 was used, the glucose oxidase content in the system was 0.0125, 0.025, 0.05, 0.1, 0.2, 0.4 and 0.5U/mL, the glucose content was 100. mu.M, the rBsDyP protein concentration was 100. mu.g/mL, and the ZEN concentration was 10. mu.g/mL, respectively, and the system without rBsDyP protein was used as a control; respectively reacting for 1h at 42 ℃, adding 1mL of methanol to terminate the reaction, and detecting the ZEN content by adopting high performance liquid chromatography.

As a result, as shown in fig. 6, as the concentration of glucose oxidase GOD increased, the degradation rate of ZEN by rbs bpyp obtained in example 1 increased, and when the concentration of GOD was 0.5U/mL, the degradation rate of ZEN by rbs bp was 75%.

Example 7 efficiency of recombinant dye decolorizing peroxidase rBsDyP to catalyze degradation of zearalenol, zearalanol, AFB2, AFM1, AFM2, AFG1 and AFG2

Respectively dissolving alpha-zearalenol, alpha-zearalanol, AFB2, AFM1, AFM2, AFG1 and AFG2 into dimethyl sulfoxide, and carrying out tests according to the following reaction systems: the degradation reaction was carried out in 1mL of buffers (pH4 and 6, 0.1M sodium citrate buffer; pH8, 0.1M sodium phosphate buffer) of different pH values, and H was added to the reaction system₂O₂The content is 100 mu M, the concentration of the rBsDyP protein prepared in example 1 is 100 mu g/mL, the concentration of zearalenol or zearalenol is 10 mu g/mL, the concentration of AFB2, AFM1, AFM2, AFG1 or AFG2 is 2 mu g/mL, and the content of ABTS is 5 mM; a system without addition of the rBsDyP protein was used as a control; after reacting for 12h at 42 ℃, 1mL of methanol is added to stop the reaction, and the content of each toxin is detected by adopting high performance liquid chromatography. The efficiency of rBsDyP to catalyze the degradation of zearalenol, zearalanol, AFB2, AFM1, AFM2, AFG1 and AFG2 is shown in table 1.

TABLE 1 efficiency of rBsDyP catalyzed degradation of different toxins

Example 8A mycotoxin biodegradation agent and a method for preparing the same

Weighing 90% of the total additive (maltodextrin and starch are mixed according to the mass ratio of 2: 1), and then mixing the carrier with the dye decolorizing peroxidase BsDyP protein prepared in the example 1 according to the proportion of 10% of the total additive to obtain the mycotoxin biodegradation agent.

Example 9A mycotoxin biodegradation agent and preparation method thereof

Weighing a carrier accounting for 9% of the total mass of the additive (maltodextrin and sucrose are mixed according to the mass ratio of 1:1), then mixing the dye decolorizing peroxidase BsDyP protein prepared in example 1 according to the mass ratio of 1% of the total mass of the additive, and finally mixing the bacillus subtilis and bacillus amyloliquefaciens mixed preparation powder according to 90% of the total mass of the additive (the mass ratio of the bacillus subtilis to the bacillus amyloliquefaciens is 1:1) to obtain the mycotoxin biodegradation agent.

Example 10A mycotoxin biodegradation agent and preparation method thereof

Firstly, 70 percent of starch and 10 percent of bacillus subtilis are mixed according to the total mass of the additive, then the dye decolorization peroxidase BsDyP protein prepared in the example 1 is mixed according to the proportion of 10 percent of the total mass of the additive, and finally the glucose oxidase is mixed according to the proportion of 10 percent of the total mass of the additive, so as to obtain the mycotoxin biodegradation agent.

Example 11A mycotoxin biodegradation agent and a method for preparing the same

The preparation method comprises the steps of mixing maltodextrin accounting for 70% of the total mass of the additive and lactobacillus acidophilus accounting for 20% of the total mass of the additive, then mixing the dye decolorization peroxidase BsDyP protein prepared in the example 1 according to the proportion of 5% of the total mass of the additive, mixing the laccase according to the proportion of 5% of the total mass of the additive, and finally mixing the syringaldehyde according to the proportion of 1% of the total mass of the additive to obtain the mycotoxin biodegradation agent.

Example 12 treatment of feed containing ZEN with mycotoxin biodegradation agent

Feed containing ZEN treated with the mycotoxin biodegrading agent described in example 11 (ZEN 4ppm) was mixed at a ratio of 0.1% and digested in vitro for 24h in simulated animal gastrointestinal fluids for degradation of ZEN in the feed.

Simulated gastric fluid: accurately weighing 2g of ZEN-containing feed, adding 2mg of degradation agent, placing into a 100mL conical flask, adding 25mL of PBS (0.1M pH6.0), adjusting pH to 6.8, and mixing. Adding 1mL of prepared amylase solution, digesting for 2h at 39 ℃ and 150 r/min. Adding 10mL of 0.2MHCl, adjusting the pH to 2.0 with 1MHCl or 1MNaOH solution, adding 1mL of freshly prepared acidic protease (50000U/g), mixing, sealing with parafilm, and culturing for 6h (150r/min) at a constant temperature of 39 ℃ by shaking.

Simulating small intestine liquid: after incubation with simulated gastric fluid for 6h, 5ml of 0.6M NaOH solution was added, the pH was adjusted to 6.8 with 1MHCl or 1M NaOH, and a freshly prepared suspension of the exogenous enzyme in the intestine (protease: amylase: lipase: 3:1:1) was added, capped with parafilm, and incubated in a constant temperature shaker at 39 ℃ for 18h (150r/min) respectively.

The degradation rate of ZEN after the end of the reaction was determined to be 94.58%.

Example 13 treatment of peanut meal containing AFB1 with a mycotoxin biodegradation agent

Peanut meal (AFB 1-1 ppm) containing AFB1 treated with the mycotoxin biodegradation agent described in example 12 was mixed at a ratio of 0.1% and digested in vitro for 24h in simulated animal gastrointestinal fluids for degrading AFB1 in feed.

Simulated gastric fluid: accurately weighing 2g of peanut meal containing AFB1, adding 2mg of degradation agent, placing into a 100mL conical flask, adding 25mL of LPBS (0.1MpH6.0), adjusting pH to 6.8, and mixing. Adding 1mL of prepared amylase solution, digesting for 2h at 39 ℃ and 150 r/min. Adding 10mL of 0.2MHCl, adjusting the pH to 2.0 with 1MHCl or 1MNaOH solution, adding 1mL of freshly prepared acidic protease (50000U/g), mixing, sealing with parafilm, and culturing for 6h (150r/min) at a constant temperature of 39 ℃ by shaking.

After the reaction, the degradation rate of AFB1 was determined to be 82.63%.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Sequence listing

<110> university of agriculture in China

<120> dye decolorization peroxidase BsDyP and application thereof in mycotoxin detoxification

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His Gln Ala Gly Ile Thr Thr Ala His Gln Thr Tyr Val Tyr Phe Ala

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Ser Ala Glu Gln Arg Asn Gln Tyr Leu Pro Pro Gln Asp Thr Gly Glu

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

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Pro Gly Phe Phe Glu Lys Asp Gly Lys Asp Arg Phe Gly Leu Lys Ser

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Lys Lys Pro Lys His Leu Ala Ala Leu Pro Ala Met Pro Asn Asp Asn

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

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Asp Asp Glu Gln Val Ala Phe His Ala Leu Arg Asn Leu Leu Asn Gln

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Ala Val Gly Thr Cys Glu Val Arg Phe Val Asn Lys Gly Phe Leu Ser

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Gly Gly Lys Asn Gly Glu Thr Pro Arg Asn Leu Phe Gly Phe Lys Asp

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Gly Thr Gly Asn Gln Ser Thr Lys Asp Asp Thr Leu Met Asn Ser Ile

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Ser Leu Lys Asp Gln Glu Asp Thr Phe Gly Arg Arg Lys Ser Ser Gly

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Asp Asn Gln Phe Ile Pro Met Leu Lys Ala Leu Ser Ala Lys Asp Ala

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Leu Asn Glu Tyr Thr Gln Thr Ile Gly Ser Ala Leu Tyr Ala Cys Pro

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Gly Lys Lys Met Ser Ala Glu Gln Arg Asn Gln Tyr Leu Pro Pro Gln

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Phe Gly Phe Gly Pro Gly Phe Phe Glu Lys Asp Gly Lys Asp Arg Phe

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gcgctgcgta acctgctaaa tcaagcggtc ggcacttgtg aggtccgttt tgtgaacaaa 660

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ggaacaggca accagagcac gaaggatgac acgttgatga actcgatcgt gtggattcag 780

tctggtgagc ctgactggat gacgggcggc acctatatgg cttttcggaa aatcaaaatg 840

ttccttgagg tatgggaccg ctcttccctc aaggaccaag aggatacctt cggccgcaga 900

aaaagctcgg gggcgccttt tgggcagaaa aaggaaacag atcccgtgaa gctgaatcaa 960

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Claims

1. The application of dye decolorizing peroxidase BsDyP with an amino acid sequence shown as SEQ ID No.1 or 2 in mycotoxin detoxification;

the mycotoxin is zearalenone, zearalenol, zearalanol, aflatoxin B1, B2, G1, G2, M1 or M2.

2. The application of the mycotoxin biodegradation agent in mycotoxin detoxification is characterized in that the mycotoxin biodegradation agent comprises dye decoloration peroxidase BsDyP or compound enzyme with an amino acid sequence shown as SEQ ID No.1 or 2, and a compatible carrier and/or mediator which are physiologically acceptable;

the complex enzyme is prepared by compounding dye decolorizing peroxidase BsDyP with an amino acid sequence shown as SEQ ID No.1 or 2 and glucose oxidase GOD;

the mediator is a natural and synthetic mediator for assisting dye decoloration peroxidase BsDyP to catalyze and degrade mycotoxin, and is one or more of syringaldehyde, acetosyringone, acetovanillone, p-coumaric acid, methyl syringate, carvacrol, thymol, cinnamaldehyde, citral, limonene, gamma terpinene, perillaldehyde, garlicin, eugenol, anethole, menthone, linalool, zingerone, isothiocyanate, 2-hydrazine-bis (3-ethyl-benzothiazole-6-sulfonic acid) ABTS, violuric acid VIO, 1-hydroxybenzotriazole HBT, 2 ', 6, 6' -tetramethylpiperidine oxide TEMPO or polyoxometallate;

3. The use according to claim 2, wherein the physiologically compatible carrier comprises maltodextrin, cyclodextrin, wheat bran, rice bran, sucrose, starch, Na₂SO₄One or more of talc, montmorillonite, oligosaccharide or yeast cell wall.

4. The use of claim 2, wherein the mycotoxin biodegrading agent further comprises a probiotic comprising one or more of enterococcus faecalis, enterococcus faecium, bacillus licheniformis, bacillus subtilis, lactobacillus plantarum, pediococcus acidilactici, bifidobacterium bifidum, enterococcus acidilactici, lactobacillus acidophilus, lactobacillus casei, lactobacillus delbrueckii subsp lactis, pediococcus pentosaceus, candida utilis, bacillus lentus, bacillus pumilus, bacillus amyloliquefaciens, bifidobacterium longum, bifidobacterium breve, bifidobacterium adolescentis, streptococcus thermophilus, lactobacillus reuteri, bifidobacterium animalis, aspergillus oryzae, lactobacillus cellobiosus, lactobacillus fermentum, or lactobacillus delbrueckii subsp bulgaricus; the mycotoxin biodegradation agent further contains at least one of lactobacillus buchneri, propionibacterium and lactobacillus paracasei when being used for silage or cattle feed; or the mycotoxin biodegradation agent also contains bacillus coagulans and/or brevibacillus laterosporus when being used for feeds of poultry, pigs and aquaculture animals.

5. The use of claim 2, wherein the mycotoxin biodegradation agent further comprises an additional enzyme; the additional enzyme comprises zearalenone lactonase, fumonisin carboxyl esterase, fumonisin aminotransferase, aminopolyol amine oxidase, aflatoxin oxidase, laccase, carboxypeptidase, aspergillus niger aspartic protease, elastase, aminopeptidase, pepsin-like protease, trypsin-like protease, bacterial protease, amylase, arabinase, arabinofuranosidase, cellulase, hemicellulase, lignin degrading enzyme, chitinase, rennin, cutinase, deoxyribonuclease, epimerase, esterase, galactosidase, glucanase, glucan lyase, endoglucanase, glucoamylase, glucosidase, glucuronidase, glucoronidase, hexose oxidase, superoxide dismutase, hydrolase, transferase, isomerase, lipolytic enzyme, lyase, xylanase, protease, xylanase, protease, xylanase, protease, hydrolase, transferase, isomerase, lipase, lipolytic enzyme, lyase, etc, One or more of mannosidase, xylanase, glutathione peroxidase, oxidoreductase, aldone reductase, pectin acetyl esterase, pectin depolymerase, pectin methylesterase, pectin lyase, manganese peroxidase, phenol oxidase, phytase, polygalacturonase, alcohol dehydrogenase, ribonuclease, transglutaminase, epoxide hydrolase, and acid phosphatase.

6. A method of degrading a mycotoxin, comprising the steps of:

treating a material containing mycotoxin with a dye decolorizing peroxidase BsDyP having an amino acid sequence shown in SEQ ID No.1 or 2 or a mycotoxin biodegradation agent as set forth in any one of claims 2 to 5;

7. The method of claim 6, wherein the material comprises one or more of a food product, a feed and raw materials thereof, a foodstuff and processing by-products thereof, or a herbal medicine.