阅读说明：本技术 一种提高染料脱色过氧化物酶的霉菌毒素降解率的方法 (Method for improving mycotoxin degradation rate of dye decolorizing peroxidase ) 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,具体涉及一种提高染料脱色过氧化物酶降解霉菌毒素效率的方法。本发明提供了两种可用于霉菌毒素玉米赤霉烯酮降解的高效染料脱色过氧化酶介体。本发明的介体能够协助链霉菌来源染料脱色过氧化物酶有效地降解霉菌毒素玉米赤霉烯酮,广泛用于食品和饲料霉菌毒素脱毒领域。(The invention relates to the technical field of agricultural biology, in particular to a method for improving the efficiency of degrading mycotoxin by using dye decolorizing peroxidase. The invention provides two efficient dye decoloration peroxidase mediators for degrading mycotoxin zearalenone. The mediator can assist streptomycete-derived dye decoloration peroxidase to effectively degrade mycotoxin zearalenone, and is widely applied to the field of food and feed mycotoxin detoxification.)

1. The method for improving the mycotoxin degradation rate of dye decolorizing peroxidase is characterized by comprising the step of using divalent manganese ions to participate in the degradation of the mycotoxin by the dye decolorizing peroxidase, wherein the mycotoxin is zearalenone, and the amino acid sequence of the dye decolorizing peroxidase is shown as SEQ ID NO. 1.

2. The method for increasing the mycotoxin degradation rate of a dye decolorization peroxidase according to claim 1, characterized in that zearalenone is degraded in a buffer solution of malonic acid-sodium malonate at a concentration of 50 mM and pH 4.0.

3. The method for increasing the mycotoxin degradation rate of a dye decolorizing peroxidase according to claim 1, characterized in that the divalent manganese ion is manganese sulfate or manganese chloride.

Technical Field

The invention relates to the technical field of agricultural biology, in particular to a method for improving the mycotoxin degradation rate of dye decolorizing peroxidase.

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 grains, feeds or foods, so that the health of the human and animal is seriously harmed. More than 400 fungal toxins have been reported, wherein the fungal toxins commonly found in feed comprise zearalenone, aflatoxin and deoxynivalenol. 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. Therefore, there is a need to establish an efficient and environmentally friendly method for detoxification of mycotoxins.

Currently, there are three main methods for mycotoxin detoxification: physical detoxification, chemical detoxification and biological detoxification. The traditional physical and chemical detoxification methods have the problems of unstable effect, large loss of nutrient components, influence on the palatability of the feed, difficulty in large-scale production and the like, and cannot be widely applied to actual production; the biological detoxification method has the characteristics of safety, high efficiency, environmental protection and the like, and has wide development and application prospects. Among them, as an environment-friendly enzyme preparation, mycotoxin degrading enzymes attract more and more attention of researchers due to the advantages of mild catalytic reaction conditions, no toxic by-products and the like.

In recent years, lignin degrading enzymes such as laccase and manganese peroxidase, as a novel mycotoxin degrading enzyme, are gradually applied to the degradation of aflatoxin, zearalenone and deoxynivalenol. According to the reports of the literature, the direct degradation of laccase and manganese peroxidase to mycotoxin is generally low, the degradation effect is often improved by means of mediator substances, and the difference of the action effects of different mediator substances is significant, for example, the degradation rate of laccase from bacillus subtilis to zearalenone can reach 100.0% under the addition of mediator methyl syringate, and the degradation rate of zearalenone to zearalenone is only 8.2% under the addition of mediator 1-hydroxybenzotriazole. It can be seen that the effect of the lignin degrading enzyme on the degradation of mycotoxins varies depending on the mediator substance.

Disclosure of Invention

The invention aims to provide a method for improving the mycotoxin degradation rate of dye decolorizing peroxidase.

The method for improving the mycotoxin degradation rate of the dye decolorizing peroxidase comprises the step of using divalent manganese ions or 1-hydroxybenzotriazole as a mediator to participate in the degradation of the mycotoxin by the dye decolorizing peroxidase, wherein the mycotoxin is zearalenone, and the dye decolorizing peroxidase is derived from streptomyces and has an amino acid sequence shown as SEQ ID NO: 1.

According to the method for improving the mycotoxin degradation rate of the dye decolorizing peroxidase, zearalenone is efficiently degraded in a buffer solution, wherein the buffer solution is a malonic acid-sodium malonate solution with the concentration of 50 mM and the pH value of 4.0.

The invention provides a reaction system for specifically improving the degradation rate of dye decolorizing peroxidase StDyP to zearalenone, which takes bivalent manganese ions or 1-hydroxybenzotriazole as a mediator to participate in the degradation of mycotoxin by the dye decolorizing peroxidase. The method of the invention can efficiently degrade the mycotoxin, has low cost and wide application range, and can be widely applied to the field of mycotoxin detoxification of foods and feeds.

Drawings

FIG. 1 shows the degradation of the mycotoxin zearalenone by a Streptomyces-derived dye decolorizing peroxidase-divalent manganese ion or 1-hydroxybenzotriazole system.

FIG. 2 shows the HPLC analysis result of the recombinant streptomyces source dye decolorization peroxidase-divalent manganese ion or 1-hydroxybenzotriazole system degradation of zearalenone.

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).

StDyP is shown in SEQ ID NO. 1, and the nucleotide sequence is shown in SEQ ID NO. 2.

Example 1 dye decolorizing peroxidaseStDyP cloning of the Gene encoding

The target gene of the invention is derived from streptomycesStreptomyces thermocarboxydusDye decolorizing peroxidase ofStDyP are provided. Sequence specific primers: 3, SEQ ID NO; 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-XbaConnecting the pCold I vectors subjected to I double enzyme digestion by a homologous recombination method, transforming a Trans I cloning host, and performing sequencing verification 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, inoculated into 50 mL LB culture solution, cultured at 37 ℃ under 200 rpm shaking for 12 h, and then transferred into 300 mL LB culture medium according to the proportion of 2%In the medium, the cells were cultured at 37 ℃ for about 4 hours (OD 600. apprxeq.0.6) with shaking at 200 rpm, followed by addition of 1 mM inducer IPTG and 10. mu.M Hemin solution to the medium, induction-culturing at 16 ℃ at 200 rpm for 12 hours, and then centrifugation 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-mediator system for 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 μ L malonic acid buffer (0.2M, pH 4.0), 20 μ L zearalenone solution (100 mg/L), 20 μ L10 mM mediator, using: manganese sulfate; 1-hydroxybenzotriazole; coumaric acid; hydroxybenzoic acid; vanillin; vanillic acid; ferulic acid; syringic acid; syringaldehyde and acetosyringone, 100. mu.L dye decolorizing peroxidase (1U/mL), 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 48 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.

As a result, as shown in fig. 1 and fig. 2, it can be seen that a part of zearalenone has been degraded, and when the mediator substance is not added to the system, the degradation rate is only 8.17%, and when the mediator substance is added to the system, the degradation rate is 27.49%; when the mediator substance 1-hydroxybenzotriazole is added, the degradation rate is 98.84%; the addition of other mediator substances such as coumaric acid, hydroxybenzoic acid, vanillin, vanillic acid, ferulic acid, syringic acid, syringaldehyde and acetosyringone has no promoting effect on the degradation of mycotoxin zearalenone by streptomyces-derived dye peroxidase. Therefore, when a mediator of divalent manganese ions is added into a degradation system, the degradation rate of zearalenone is improved by 3.36 times; the degradation rate of zearalenone is improved by 12.10 times by adding a mediator 1-hydroxybenzotriazole; the addition of the mediator substance of divalent manganese ions or 1-hydroxybenzotriazole obviously improves the degradation rate of the dye decolorizing peroxidase on zearalenone.

Sequence listing

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

<120> a method for increasing mycotoxin degradation rate of dye decolorizing peroxidase

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

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

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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

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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

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

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

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

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

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

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

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