Zearalenone hydrolase ZHRnZ, coding gene and application of zearalenone hydrolase ZHRnZ

Abstract

The invention belongs to the technical field of agricultural biology, and particularly relates to zearalenone hydrolase ZHRnZ as well as a coding gene, a preparation method and application thereof. The amino acid sequence of the zearalenone hydrolase ZHRnZ provided by the invention is as shown in SEQ ID NO.1, and the nucleotide sequence of the coding gene of the zearalenone hydrolase ZHRnZ is as shown in SEQ ID NO.2. The hydrolase provided by the invention has relatively strong degradation capability on zearalenone, derivatives of zearalenone and macrolide antibiotics, and is high in degradation speed. The method has a huge application prospect in the fields of degradation of feed, food, biomass energy, Chinese herbal medicine mycotoxin and antibiotics.

Description

Technical Field

[0001] The invention belongs to the field of agricultural biotechnology, and specifically relates to a zearalenone hydrolase ZHRnZ and a coding gene and application thereof, in particular to the application in the detoxification of zearalenone and its derivatives and macrolide antibiotics. Background Art

[0002] Zearalenone (ZEN) is a toxic secondary metabolite mainly produced by Fusarium fungi. The molecular formula of zearalenone is C18H22O5. It is a lactone structure substance of phenolic dihydroxybenzoic acid, similar to estradiol. Therefore, zearalenone and its derivatives can competitively bind to estrogen receptors in animals and exert estrogen-like effects. During metabolism in animals, zearalenone will be converted into derivatives such as α-zearalenol, β-zearalenol, α-zearalenol, and β-zearalenol, and their estrogenic activities are 30 times, 0.44 times, 18 times, and 3.5 times that of zearalenone, respectively.

[0003] Corn, wheat and other crops often suffer from Fusarium diseases, and the toxins such as zearalenone produced by the fungus are widely present in moldy crops, endangering food and feed safety. Long-term intake of zearalenone-contaminated feed by female animals can lead to estrogen syndrome, estrous cycle disorders, decreased reproductive capacity, and increased abortion rates; long-term intake of zearalenone-contaminated feed by male animals can lead to decreased sperm activity and semen quality. The use of biodegradation to convert zearalenone and its derivatives into non-toxic or low-toxic products has the advantages of high efficiency, environmental protection, and little impact on feed resale. At present, research mainly focuses on the hydrolysis of lactone ring opening pathways. In 2002, Takahashi-Ando et al. first isolated a lactone hydrolase ZHD101 that can degrade ZEN from Clonostachys rosea IFO 7063, and subsequently a variety of lactone hydrolases that can degrade zearalenone were gradually reported.

[0004] However, existing zearalenone hydrolases still have many shortcomings: the optimal pH for zearalenone degradation is 8-9.5, and the catalytic activity cannot be fully exerted in the digestive tract environment; the temperature tolerance is poor, and it cannot withstand the high-temperature pelleting of feed; in particular, there are few reports on this type of enzyme in the degradation of macrolide antibiotics. Therefore, it is urgent to expand and supplement the resources of zearalenone hydrolases and explore zearalenone hydrolases with good thermal stability and high efficiency under neutral or acidic conditions. Summary of the invention

[0005] To solve the above technical problems, the present invention provides a zearalenone hydrolase ZHRnZ, a coding gene, a recombinant vector and a recombinant strain containing the gene, a method for expressing the zearalenone hydrolase, and the use of the enzyme in degrading zearalenone and its derivatives and macrolide antibiotics.

[0006] To achieve the above objectives, in a first aspect, the present invention provides a corn zearalenone hydrolase ZHRnZ, wherein the amino acid sequence of the corn zearalenone hydrolase ZHRnZ is shown in SEQ ID NO.1.

[0007] In a second aspect, the present invention also provides a gene encoding the above-mentioned zearalenone hydrolase ZHRnZ, as shown in SEQ ID NO.2.

[0008] In a third aspect, the present invention also provides a recombinant expression vector comprising the above-mentioned zearalenone hydrolase ZHRnZ encoding gene.

[0009] In a fourth aspect, the present invention also provides a recombinant strain comprising the above-mentioned zearalenone hydrolase ZHRnZ encoding gene, wherein the recombinant strain is transformed by a recombinant vector comprising the ZHRnZ encoding gene, and the recombinant strain can be Escherichia coli, Bacillus subtilis, Bacillus licheniformis, Lactococcus lactis, Lactobacillus plantarum, Lactobacillus casei, Pichia pastoris, Saccharomyces cerevisiae, Kluyveromyces lactis, Aspergillus oryzae, Aspergillus niger and Trichoderma reesei.

[0010] In a fifth aspect, the present invention also provides a method for preparing the above-mentioned zearalenone hydrolase ZHRnZ. The method comprises: (1) transforming the recombinant expression vector pET-28a-ZHRnZ containing the ZHRnZ encoding gene into Escherichia coli BL21 (DE3) to obtain a recombinant strain; (2) culturing the recombinant strain to induce the expression of the ZHRnZ protein.

[0011] In a sixth aspect, the present invention also provides an application of the above-mentioned zearalenone hydrolase ZHRnZ in the efficient degradation of zearalenone and its derivatives.

[0012] In a seventh aspect, the present invention also provides an application of the above-mentioned zearalenone hydrolase ZHRnZ in the efficient degradation of macrolide antibiotics.

[0013] In an eighth aspect, the present invention also provides an additive capable of degrading zearalenone and its derivatives and macrolide antibiotics.

[0014] In a ninth aspect, the present invention also provides the use of the above-mentioned additive in the biodegradation of zearalenone and its derivatives and macrolide antibiotics.

[0015] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0016] The corn zearalenone hydrolase ZHRnZ provided by the present invention has an amino acid sequence as shown in SEQ ID NO.1:

[0017] MRRTRGTTTTAEGITWYYEQEGTGPDVVLVPDGLGECQMFDGALSRLAARGFRATTFDMPGMSRSAAAPRAAYEDVTAQKLAAYVASVTAALGIGVDGAPPAAFWGCSSGGAAVLALVAGYPERVRCALVHEVPTYH MEDLVSARELGDDDAVAASLAASVPAGSYARGGDLRAWFALGGDAHARLWRNYPRWARGYPRTIPPSSPTAPEDLARRPLYWTVGADTPASRFLDNVVTAAKNGIDVGCLPGMHFPYVSHPDAFVKHVADIVSKHL

[0018] The enzyme consists of 272 amino acids, no signal peptide sequence is predicted, and the theoretical molecular weight of the protein is 28.8 kDa.

[0019] The gene encoding the above-mentioned zearalenone hydrolase ZHRnZ of the present invention has a coding gene sequence as shown in SEQ ID NO.2:

[0020] ATGCGCACCCGCGGCACCACGACCACCGCGGAAGGCATTACCTGGTATTATGAACAAGAAGGCACCGGCCCGGATGTGGTGCTGGTGCCGGATGGCCTGGGCGAATGTCAGATGTTTGATGGCGCCTGAGCCGCCTGGCGGCGCGCGGCTTTCGCGCGACCACCTTTGATATGCCGGGCATGAGCCGCAGCGCGGCCGCGCCG CGCGCGGCGTATGAAGATGTGACCGCGCAAAAGCTGGCGGCCTATGTGGCGAGCGTTACGGCGGCGTTAGGCATTGGCGTGGACGGCGCCGCCGGCGGCGTTTTGGGGCTGCAGCAGCGGCGGTGCGGCGGTTCTGGCGCTGGTGGCGGGTTATCCGGAACGCGTGCGCTGCGCGCTGGTGCATGAAGTGCCGACCTATCAT ATGGAAGATTTAGTGAGTGCGCGCGAACTGGGTGATGACGACGCGGTGGCGGCGAGCTTAGCCGCGAGTGTGCCGGCCGGCAGCTACGCGCGCGGTGGTGATTTACGCGCGTGGTTTGCTGGGCGGTGATGCGCACGCCCGCTTATGGCGCAATTATCCGCGCTGGGCGCGCGGCTACCCGCGCACGATCCCGCCAAGCAGC CCAACCGCGCCGGAAGACCTGGCGCGCCGTCCGCTGTATTGGACCGTGGGCGCGGATACCCCGGCGAGCCGCTTTCTGGATAACGTGGTGACCGCCGCGAAAAACGGCATTGATGTGGGCTGCCTGCCGGGCATGCATTTTCCGTATGTGAGCCATCCGGATGCGTTTGTGAAACATGTGGCGGATATTGTGAGCAAACATCTG

[0021] The present invention also provides a recombinant vector comprising the above-mentioned zearalenone hydrolase ZHRnZ, preferably pET-28a-ZHRnZ.

[0022] The present invention also provides a recombinant strain comprising the above-mentioned zearalenone hydrolase ZHRnZ, preferably a recombinant strain BL21(DE3) / pET-28a-ZHRnZ.

[0023] The present invention also provides a method for preparing zearalenone hydrolase ZHRnZ, comprising the following steps:

[0024] (1) transforming a host cell with a recombinant expression vector comprising a gene encoding zearalenone hydrolase ZHRnZ to obtain a recombinant strain;

[0025] (2) culturing the recombinant strain to induce the expression of zearalenone hydrolase ZHRnZ protein;

[0026] (3) The purified zearalenone hydrolase ZHRnZ was obtained by nickel ion affinity chromatography.

[0027] The present invention also provides the use of the above-mentioned zearalenone hydrolase ZHRnZ in degrading zearalenone and its derivatives and macrolide antibiotics, including applications in feed industry, food industry, biomass energy and Chinese herbal medicine processing.

[0028] The present invention also provides an additive for degrading zearalenone and its derivatives and macrolide antibiotics, comprising zearalenone hydrolase ZHRnZ and a physiologically acceptable carrier thereof. The content of zearalenone hydrolase ZHRnZ is 1%-10%; the physiologically acceptable carrier is selected from one or more of wheat, rice, oat bran, rice bran, sucrose, starch, cornmeal, maltodextrin, cyclodextrin, bentonite, montmorillonite, oligosaccharide or yeast cell wall.

[0029] The present invention also provides the application of the additive, especially in the fields of feed industry, food industry, biomass energy and Chinese herbal medicine processing, etc., in the biodegradation of zearalenone and its derivatives, and macrolide antibiotics.

[0030] Advantages and beneficial effects of the present invention:

[0031] The present invention provides a zearalenone hydrolase ZHRnZ, a coding gene, an additive and an application thereof, and in particular relates to an application thereof in the degradation of zearalenone and its derivatives and macrolide antibiotics. Experiments have confirmed that the zearalenone hydrolase ZHRnZ has a strong degradation ability for zearalenone and its derivatives and macrolide antibiotics, and can completely degrade 10 μg / mL of substrate in 20 minutes, and has a very broad application prospect in production practice. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following briefly introduces the drawings required for use in the embodiments or the prior art description:

[0033] Figure 1Shown is the SDS-PAGE electrophoresis analysis of the expression product of the recombinant plasmid pET-28a-ZHRnZ after purification;

[0034] Figure 2 Shown are the results of HPLC analysis of ZEN degradation by ZHRnZ protein (A is the ZEN standard control group, B is the ZHRnZ protein-treated ZEN group);

[0035] Figure 3 Shown is the degradation of ZHRnZ protein to different concentrations of ZEN at different reaction times;

[0036] Figure 4 Shown is the degradation of ZEN by ZHRnZ protein under different pH conditions;

[0037] Figure 5 Shown is the degradation of ZEN by ZHRnZ protein under different temperature conditions;

[0038] Figure 6 Shown is the stability effect diagram of ZHRnZ protein at different temperatures;

[0039] Figure 7 Shown is the stability effect diagram of ZHRnZ protein at different pH;

[0040] Figure 8 The figure shows the degradation of zearalenone derivatives by ZHRnZ protein;

[0041] Fig. 9 Shown is the degradation of macrolide antibiotics by ZHRnZ protein. DETAILED DESCRIPTION

[0042] The present invention discloses a corn zearalenone hydrolase ZHRnZ, a coding gene and its application. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It should be particularly noted that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0043] The present invention will be further described below in conjunction with the accompanying drawings and specific examples. Unless otherwise specified, 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.

[0044] The main experimental materials and reagents used in the examples of the present invention are:

[0045] The E. coli expression vector pET-28a, cloning strain E. coli DH5α, and expression strain E. coli BL21 (DE3) were all purchased from Invitrogen. Restriction endonucleases and DNA ligases were purchased from NEB, mycotoxin standards were purchased from Sigma, and other reagents were domestic analytical grade.

[0046] Example 1 Acquisition and expression of corn zearalenone hydrolase ZHRnZ

[0047] 1. Artificial synthesis of gene sequences

[0048] The optimized nucleotide sequence shown in SEQ ID NO.1 was commissioned to Suzhou Jinweizhi Biotechnology Co., Ltd. for artificial gene synthesis according to conventional techniques in the art, and the gene was inserted into the plasmid vector pUC19.

[0049] 2. Amplification of gene sequences and construction of recombinant expression vectors

[0050] The nucleotide sequence shown in SEQ ID NO.1 was amplified using primers ZHRnZ-F and ZHRnZ-R, and named as ZHRnZ-DNA fragment. The reaction conditions were:

[0051] DNA template 1μL Upstream primer P1 2μL Downstream primer P2 2μL 2×Pfu PCR Mix 25μL <![CDATA[ddH2O2]]> 20μL Total volume 50μL

[0052] The amplification conditions were as follows: 95°C pre-denaturation for 5 min; 95°C denaturation for 30 s; 50°C annealing for 30 s, 72°C extension for 1 min 30 s for 30 cycles; 72°C complete extension for 10 min. The PCR amplification products were subjected to 1% agarose gel electrophoresis, and the PCR products were recovered using an agarose gel DNA recovery kit.

[0053] The plasmid pET-28a was double-digested with BamHI and XhoI, and the enzyme digestion system was as follows:

[0054] pET-28a plasmid 30μL B H 1μL XOt 1μL 10×CutSmart Buffer 5μL <![CDATA[ddH2O2]]> 13μL Total volume 50μL

[0055] The digestion conditions were: 37°C water bath for 30 min. The digestion products were subjected to 1% agarose gel electrophoresis, and the plasmid digestion fragments were recovered using an agarose gel DNA recovery kit.

[0056] The ZHRnZ-DNA fragment recovered by the above-mentioned enzyme digestion and the double-enzyme-digested plasmid were ligated overnight at 16°C using T4 DNA ligase; the ligation product was transformed into Escherichia coli competent cells BL21 (DE3), and screened on an LB plate with ampicillin resistance (final ampicillin concentration of 100 μg / mL), and the positive transformants were picked, the transformation plasmid was extracted, and single and double enzyme digestion verification and sequencing were performed to determine that the correct recombinant strain BL21 (DE3) / pET-28a-ZHRnZ was constructed.

[0057] Ampicillin-resistant LB solid plate: weigh 1g sodium chloride, 1g tryptone, 0.5g yeast extract, 1.5g agar powder, dissolve in 100mL ultrapure water, sterilize at 121℃, cool to 50-60℃, add ampicillin to a final concentration of 100μg / mL, pour into disposable sterile plates, cool and set aside.

[0058] 3. Inducible expression of recombinant strain BL21(DE3) / pET-28a-ZHRnZ

[0059] The positive recombinant bacteria BL21(DE3) / pET-28a-ZHRnZ prepared in step 2 above were inoculated into 5 mL of liquid LB medium for activation overnight, and then transferred into 500 mL of liquid LB medium at a ratio of 1:100, and cultured at 180 rpm and 37°C for 3-4 h. The OD 600 , wait for OD 600 When the pH value reached 0.4-0.8, isopropyl-β-D-thiogalactopyranoside (IPTG) was added to a final concentration of 0.4 mmol / L, and the culture was carried out at 16°C and 120 rpm for 16-20 h.

[0060] Liquid LB medium: weigh 10 g sodium chloride, 10 g tryptone, and 5 g yeast extract, dissolve in 1 L ultrapure water, sterilize at 121°C and set aside.

[0061] 1 mol / L IPTG solution: Weigh 2.38 g IPTG, dissolve it in ultrapure water and make up to 10 mL, filter it with a 0.22 μm sterile filter membrane to sterilize it, and store it at -20°C for later use.

[0062] 4. Purification of Zearalenone Hydrolase ZHRnZ Protein

[0063] After the induction is completed, the fermentation broth was centrifuged at 4°C and 4000rpm for 30min, the supernatant was discarded and the bacteria were collected; the bacteria were resuspended with 25mMTris-HCl (pH8.0) buffer, centrifuged at 4°C and 4000rpm for 30min, the supernatant was discarded, and the bacteria were washed three times. After the washing, the bacteria were resuspended with 100mL of Binding Buffer and ultrasonically disrupted three times under the conditions of: power 270W, working for 3s and resting for 5s, each time for 10min. After the cells were disrupted, centrifuged at 4°C and 12000rpm for 10min, the supernatant was collected and filtered through a 0.45μm filter membrane. According to the recombinase fusion expression of the His-tag tag at the N-terminus, Ni 2+ -NTA prepacked columns combined with NGC TMThe protein was purified using the fully automated protein purification system. The steps of equilibration, loading, and elution were described in the Qiagen user manual. The purification results of the target protein were analyzed using SDS-PAGE electrophoresis. Figure 1 As shown, the recombinant enzyme ZHRnZ has a single and obvious band in lane 2, and the molecular weight is about 28.8 kDa, which is consistent with the theoretical molecular weight, indicating that the recombinant enzyme ZHRnZ is successfully expressed and purified.

[0064] 25mM Tris-HCl buffer: weigh 3.025g Tris, dissolve in 500mL ultrapure water, adjust pH to 8.0 with hydrochloric acid, and make up to 1L.

[0065] Binding Buffer (25mM Tris, 150mM NaCl, 5mM Imidazole): weigh 3.025g Tris, 8.762g NaCl, take 2.5mL 2M Imidazole, dissolve in 500mL, adjust pH to 8.0 with hydrochloric acid, and make up to 1L. Filter with a 0.45μm filter membrane to remove impurities before use.

[0066] Elution Buffer (25mM Tris, 150mM NaCl, 500mM Imidazole): weigh 3.025g Tris, 8.762g NaCl, take 250mL 2M Imidazole, dissolve in 500mL, adjust pH to 8.0 with hydrochloric acid, and make up to 1L. Filter with a 0.45μm filter membrane to remove impurities before use.

[0067] Example 2 Detection of the degradation activity of zearalenone by zearalenone hydrolase ZHRnZ

[0068] The protein concentration of the zearalenone hydrolase ZHRnZ prepared in Example 1 was determined to be 9.4 mg / mL. It was diluted ten times with 25 mM Tris-HCl buffer and the diluted enzyme solution was used for subsequent experiments.

[0069] Zearalenone was dissolved in methanol to prepare a 500 μg / mL mother solution, and the experiment was carried out according to the following 200 μL reaction system: 190.7 μL Tris-HCl buffer (25 mM, pH 7.5), 5.3 μL zearalenone hydrolase ZHRnZ (final concentration of 25 μg / mL), 4 μL zearalenone solution (500 μg / mL, final concentration of 10 μg / mL). The system without ZHRnZ protein was used as a control. The reaction was carried out at 37°C for 15 min, and 200 μL methanol (HPLC grade) was added to terminate the reaction. The zearalenone content in the sample was detected by high performance liquid chromatography (HPLC), and the activity of zearalenone hydrolase ZHRnZ in degrading ZEN was analyzed according to the change of ZEN content.

[0070] The conditions for HPLC detection of ZEN were as follows: chromatographic column: Agilent C18 chromatographic column (4.6 mm × 150 mm × 5 μm); column temperature: 30°C; mobile phase: acetonitrile-water (60:40); flow rate: 1 mL / min; injection volume: 20 μL; fluorescence detector detection wavelength: λex = 274 nm, λem = 440 nm.

[0071] Figure 2 Shown is the HPLC chromatogram of the degradation of ZEN by the recombinant corn zearalenone hydrolase ZHRnZ, A is the ZEN control group, B is the ZEN group treated with ZHRnZ protein. As shown in the figure, the control group sample has a strong ZEN absorption peak at the retention time RT = 5.831min, while no ZEN absorption peak is detected in the ZHRnZ protein treated group at the retention time. The results show that the recombinant corn zearalenone hydrolase ZHRnZ has the activity of efficiently degrading ZEN, and the degradation rate of ZEN is 100%.

[0072] Example 3 Degradation activity of zearalenone hydrolase ZHRnZ on different concentrations of ZEN under different reaction time conditions

[0073] In order to select the appropriate ZEN concentration and reaction time, three ZEN concentration levels of 2μg / mL, 10μg / mL, and 20μg / mL and six time levels of 2, 5, 10, 15, 30, and 60min were set. The reaction system was 200μL: 190.7μL Tris-HCl buffer (25mM, pH7.5), 5.3μL zearalenone hydrolase ZHRnZ (final concentration of 25μg / mL), and 4μL zearalenone solution (final concentration of 2, 10, and 20μg / mL). The reaction was carried out at 37℃ for 15min, and 200μL methanol (HPLC grade) was added to terminate the reaction. The ZEN content in the sample was detected by high performance liquid chromatography (HPLC). The results are shown in Figure 2. Figure 3 shown.

[0074] Figure 3 The results showed that the zearalenone hydrolase ZHRnZ has the activity of efficiently degrading ZEN. Under the conditions of 37℃ and pH8.0, 2μg / mL ZEN can be degraded by more than 80% in 5 minutes; for 10μg / mL and 20μg / mL ZEN, the degradation rate is more than 50% in 15 minutes and more than 80% in 60 minutes. The reaction conditions for subsequent experiments were selected as 10μg / mL ZEN and 15 minutes of reaction.

[0075] Example 4 Effect of pH on the activity of zearalenone hydrolase ZHRnZ in degrading ZEN

[0076] To test the effect of reaction system pH on the degradation activity of ZEN by zearalenone hydrolase ZHRnZ, ten pH levels were set: 3, 4, 5, 6, 7, 8, 9, 9.5, 10, and 11. The reaction system was 200 μL: 190.7 μL of different pH buffers (pH 3.0-6.0: 50 mM sodium dihydrogen phosphate-citrate buffer; pH 7.0-10.0: 50 mM Tris-HCl buffer; pH 11.0: 50 mM sodium hydroxide-glycine buffer), 5.3 μL of zearalenone hydrolase ZHRnZ (final concentration of 25 μg / mL), and 4 μl of ZEN (500 μg / mL, final concentration of 10 μg / mL). The reaction was carried out at 37°C for 15 min, and 200 μL of methanol (HPLC grade) was added to terminate the reaction. The ZEN content in the sample was detected by high performance liquid chromatography (HPLC), and the degradation rate was calculated. The degradation rate under the optimal pH condition was defined as 100%, and the relative activity under different pH conditions was calculated.

[0077] The results are as follows Figure 4 As shown, when ZHRnZ uses ZEN as substrate, the optimum pH is 9, and its relative activity is maintained above 63.5% in the pH range of 6-10.

[0078] Example 5 Effect of temperature on the activity of zearalenone hydrolase ZHRnZ in degrading ZEN

[0079] To test the effect of temperature on the degradation activity of ZEN by zearalenone hydrolase ZHRnZ, nine temperature levels of 30, 37, 40, 42, 45, 47, 50, 60, and 70°C were set. The reaction system was 200 μL: 190.7 μL pH 9.0 buffer (50 mM Tris-HCl), 5.3 μL zearalenone hydrolase ZHRnZ (final concentration of 25 μg / mL), and 4 μL ZEN solution (final concentration of 10 μg / mL). The reaction was carried out at 37°C for 15 min, and 200 μL methanol (HPLC grade) was added to terminate the reaction. The ZEN content in the sample was detected by high performance liquid chromatography (HPLC), and the degradation rate was calculated. The degradation rate under the optimal temperature condition was defined as 100%, and the relative activity under different temperature conditions was calculated.

[0080] The results are as follows Figure 5 As shown, when ZHRnZ uses ZEN as substrate, the optimum temperature is 45°C, and its relative activity is maintained above 73.2% in the range of 30-50°C.

[0081] Example 6 Temperature stability of zearalenone hydrolase ZHRnZ

[0082] To test the temperature stability of zearalenone hydrolase ZHRnZ, the residual activity of ZHRnZ recombinant protein in degrading ZEN was determined after being treated at different temperatures for different times. Three temperature levels of 30, 40, and 50°C and six treatment time levels of 0, 2, 5, 10, 15, and 20 min were set. After being treated at different temperatures for different times, the samples were placed in an ice bath for 1 min, and the residual activity was determined under the optimal conditions. The reaction system was 200 μL: 190.7 μL pH 9.0 buffer (50 mM Tris-HCl), 5.3 μL zearalenone hydrolase ZHRnZ (final concentration of 25 μg / mL), and 4 μL ZEN solution (final concentration of 10 μg / mL). The reaction was carried out at 37°C for 15 min, and 200 μL methanol (HPLC grade) was added to terminate the reaction. The ZEN content in the sample was detected by high performance liquid chromatography (HPLC), and the degradation rate was calculated. The degradation rate of 0 min treatment was defined as 100%, and the relative activity under different conditions was calculated.

[0083] The results are as follows Figure 6 As shown, temperature has a great influence on the stability of zearalenone ZHRnZ. After treatment at 30℃ for 15 minutes, 49.6% relative activity remained, after treatment at 40℃ for 5 minutes, 48.8% relative activity remained, and after treatment at 50℃ for 2 minutes, it was completely inactivated.

[0084] Example 7 pH stability of zearalenone hydrolase ZHRnZ

[0085] To test the pH stability of zearalenone hydrolase ZHRnZ, the residual activity of ZHRnZ recombinant protein in degrading ZEN was determined after being treated in different pH buffers for different time periods. Six pH levels of 4, 5, 6, 7, 8, and 9 were set, and five treatment time levels of 1h, 6h, 12h, 24h, and 48h were set. After being treated for different time periods under different pH conditions, the residual activity was determined under the optimal conditions. The reaction system was 200μL: 190.7μL pH9.0 buffer (50mM Tris-HCl), 5.3μL zearalenone hydrolase ZHRnZ (final concentration of 25μg / mL), and 4μL ZEN solution (final concentration of 10μg / mL). The reaction was carried out at 37℃ for 15min, and 200μL methanol (HPLC grade) was added to terminate the reaction. The ZEN content in the sample was detected by high performance liquid chromatography (HPLC), and the degradation rate was calculated. The degradation rate of 0min treatment was defined as 100%, and the relative activity under different conditions was calculated.

[0086] The results are as follows Figure 7 As shown, zearalenone ZHRnZ has good pH stability in an alkaline environment, and the enzyme still has 98.8% residual activity after being treated in pH = 9 buffer for 48 hours.

[0087] Example 8 Degradation activity of zearalenone hydrolase ZHRnZ on zearalenone derivatives

[0088] α-Zearalenol (α-ZOL), β-Zearalenol (β-ZOL) and α-Zeranol (α-ZAL) were dissolved in methanol to prepare 500 μg / mL stock solution, and the experiment was carried out according to the following 200 μL reaction system: 190.7 μL pH 9.0 buffer (50 mM Tris-HCl), 5.3 μL zearalenone hydrolase ZHRnZ (final concentration of 25 μg / mL), 4 μL toxin stock solution (500 μg / mL, final concentration of 10 μg / mL). The system without ZHRnZ protein was used as a control. The reaction was carried out at 37℃ for 15 minutes, and 200 μL methanol (HPLC grade) was added to terminate the reaction. The content of zearalenone derivatives in the sample was detected by high performance liquid chromatography (HPLC), and the degradation rate was calculated.

[0089] The results are as follows Figure 8 As shown, ZHRnZ protein can degrade more than 90% of zearalenone derivatives in the system by reacting with 10 μg / mL α-ZOL, β-ZOL or α-ZAL for 15 min.

[0090] Example 9 Degradation activity of zearalenone hydrolase ZHRnZ on macrolide antibiotics

[0091] Dissolve tylosin and erythromycin in methanol to prepare 500μg / mL and 2500μg / mL stock solutions, respectively. Perform the experiment in the following 200μL reaction system: 190.7μL pH 9.0 buffer (50mM Tris-HCl), 5.3μL zearalenone hydrolase ZHRnZ (final concentration 25μg / mL), 4 tylosin solution (500μg / mL, final concentration 10μg / mL) or 4μL erythromycin solution (2500μg / mL, final concentration 50μg / mL). The system without ZHRnZ protein was used as a control. React at 37℃ for 15min, add 200μL methanol (HPLC grade) to terminate the reaction, and use high performance liquid chromatography (HPLC) to detect the content of erythromycin or tylosin in the sample, and calculate the degradation rate.

[0092] The results showed that ZHRnZ protein could degrade 55.9% of tylosin in the system by reacting with 10 μg / mL tylosin for 1 h, and could degrade 68.9% of erythromycin in the system by reacting with 50 μg / mL erythromycin for 1 h.

[0093] The liquid chromatography detection conditions of erythromycin are as follows: chromatographic column: Agilent C18 chromatographic column (4.6mm×150mm×5μm); column temperature: 25°C; mobile phase: 0.1mol / L ammonium dihydrogen phosphate (triethylamine adjusted to pH 5.9): acetonitrile = 6:4; flow rate: 1mL / min; injection volume: 20μL; UV detector detection wavelength: 210nm.

[0094] The liquid chromatography conditions of tylosin are as follows: chromatographic column: Agilent C18 chromatographic column (4.6 mm×150 mm×5 μm); column temperature: 30°C; mobile phase: acetonitrile: methanol: 0.025 mol / L ammonium dihydrogen phosphate = 25:30:5; flow rate: 0.6 mL / min; injection volume: 20 μL; detection wavelength of ultraviolet detector: λex = 290 nm.

[0095] Example 10 An additive and a method for preparing the same

[0096] Weigh 95% of the total mass of the additive as a carrier (wheat: starch: bentonite mixed in a mass ratio of 2:1:2), and then mix zearalenone hydrolase ZHRnZ in a ratio of 5% of the total mass of the additive to obtain the additive.

[0097] Example 11 An additive and a method for preparing the same

[0098] Weigh 96% of the total mass of the additive as a carrier (rice: oat bran: oligosaccharide mixed in a mass ratio of 5:4:1), and then mix zearalenone hydrolase ZHRnZ in a ratio of 4% of the total mass of the additive to obtain the additive.

[0099] Example 12 An additive and a method for preparing the same

[0100] The additive was obtained by weighing a carrier (rice bran: sucrose: montmorillonite mixed in a mass ratio of 3:1:1) which was 97% of the total mass of the additive, and then mixing zearalenone hydrolase ZHRnZ in a ratio of 3% of the total mass of the additive.

[0101] Example 13 An additive and its preparation method

[0102] Weigh 98% of the total mass of the additive as a carrier (cornmeal: cyclodextrin: yeast cell wall mixed in a mass ratio of 5:3:2), and then mix zearalenone hydrolase ZHRnZ in a ratio of 2% of the total mass of the additive to obtain the additive.

[0103] Example 14 A method for degrading zearalenone

[0104] The feed containing ZEN was treated with the additive described in Example 10, and the additive was mixed with naturally moldy feed (ZEN = 10 μg / mL) at a ratio of 0.1%, and digested in simulated animal gastrointestinal fluid in vitro for 24 hours to degrade ZEN in the feed.

[0105] Simulated gastric fluid: Accurately weigh 2g of feed containing ZEN, add 2mg of additives, put into a 100mL conical flask, add 25mL PBS (0.1M pH6.0), adjust pH to 6.8, mix well. Add 1mL of prepared amylase solution, digest at 39℃, 150r / min for 2h. Add 10mL 0.2M HCl, adjust pH to 2.0 with 1M HCl or 1M NaOH solution, add 1mL of freshly prepared acid protease (50000U / g), mix well, seal with parafilm, and culture at 39℃ constant temperature shaker for 6h (150r / min).

[0106] Simulated small intestinal fluid: After incubating in simulated gastric fluid for 6 hours, add 5 mL of 0.6 M NaOH solution, adjust the pH to 6.8 with 1 M HCl or 1 M NaOH, and then add freshly prepared intestinal exogenous enzyme suspension (protease: amylase: lipase = 3:1:1), seal with parafilm, and incubate in a 39°C constant temperature shaker for 18 hours (150 r / min).

[0107] After the reaction was completed, the degradation rate of ZEN in the feed was determined to be 92.35%.

[0108] Example 15 A method for degrading zearalenone and its derivatives

[0109] The ZEN, α-ZOL, β-ZOL and α-ZAL in DDGS, a by-product of industrial ethanol processing, were treated with the additive described in Example 11. The additive was mixed with DDGS, a by-product of industrial ethanol processing (ZEN = 10 μg / mL) at a ratio of 0.1% and digested in simulated animal gastrointestinal fluid in vitro for 24 hours.

[0110] Simulated gastric fluid: Accurately weigh 2g of moldy DDGS, add 2mg of additives, put into a 100mL conical flask, add 25mL PBS (0.1M pH6.0), adjust the pH to 6.8, and mix well. Add 1mL of prepared amylase solution, digest at 39℃, 150r / min for 2h. Add 10mL 0.2M HCl, adjust the pH to 2.0 with 1M HCl or 1M NaOH solution, add 1mL of freshly prepared acid protease (50000U / g), mix well, seal with parafilm, and culture at 39℃ constant temperature shaker for 6h (150r / min).

[0111] Simulated small intestinal fluid: After incubating in simulated gastric fluid for 6 hours, add 5 mL of 0.6 M NaOH solution, adjust the pH to 6.8 with 1 M HCl or 1 M NaOH, and then add freshly prepared intestinal exogenous enzyme suspension (protease: amylase: lipase = 3:1:1), seal with parafilm, and incubate in a 39°C constant temperature shaker for 18 hours (150 r / min).

[0112] After the reaction, the degradation rates of ZEN, α-ZOL, β-ZOL and α-ZAL in DDGS were determined to be 90.23%, 95.16%, 93.68% and 92.15%, respectively.

[0113] Example 16 A method for degrading zearalenone and its derivatives

[0114] ZEN, α-ZOL, β-ZOL and α-ZAL in oat bran were treated with the additive described in Example 12. The additive was mixed with oat bran (ZEN = 8 μg / mL) at a ratio of 0.1% and digested in simulated animal gastrointestinal fluid in vitro for 24 h.

[0115] Simulated gastric fluid: Accurately weigh 2g of oat bran (ZEN = 8μg / mL), add 2mg of additives, put into a 100mL conical flask, add 25mL PBS (0.1M pH6.0), adjust pH to 6.8, and mix well. Add 1mL of prepared amylase solution, digest at 39℃, 150r / min for 2h. Add 10mL 0.2M HCl, adjust pH to 2.0 with 1M HCl or 1M NaOH solution, add 1mL of freshly prepared acid protease (50000U / g), mix well, seal with parafilm, and culture at 39℃ constant temperature shaker for 6h (150r / min).

[0116] Simulated small intestinal fluid: After incubating in simulated gastric fluid for 6 hours, add 5 mL of 0.6 M NaOH solution, adjust the pH to 6.8 with 1 M HCl or 1 M NaOH, and then add freshly prepared intestinal exogenous enzyme suspension (protease: amylase: lipase = 3:1:1), seal with parafilm, and incubate in a 39°C constant temperature shaker for 18 hours (150 r / min).

[0117] After the reaction, the degradation rates of ZEN, α-ZOL, β-ZOL and α-ZAL in oat bran were determined to be 91.88%, 96.36%, 92.15% and 94.39%, respectively.

[0118] Example 17 A method for degrading macrolide antibiotics

[0119] The additive described in Example 13 was used to treat tylosin and erythromycin in the Chinese herbal medicine. The mother solutions of tylosin and erythromycin were added to the Chinese herbal medicine. The additive and the Chinese herbal medicine (tylosin = 10 μg / mL; erythromycin = 15 μg / mL) were mixed at a ratio of 0.1%, and digested in simulated animal gastrointestinal fluid in vitro for 24 hours.

[0120] Simulated gastric fluid: Accurately weigh 2g of Chinese herbal medicine (tylosin = 10μg / mL; erythromycin = 15μg / mL), add 2mg of additives, put into a 100mL conical flask, add 25mL PBS (0.1M pH6.0), adjust the pH to 6.8, and mix well. Add 1mL of prepared amylase solution, digest at 39℃, 150r / min for 2h. Add 10mL 0.2M HCl, adjust the pH to 2.0 with 1M HCl or 1M NaOH solution, add 1mL of freshly prepared acid protease (50000U / g), mix well, seal with parafilm, and culture at 39℃ constant temperature shaker for 6h (150r / min).

[0121] Simulated small intestinal fluid: After incubating in simulated gastric fluid for 6 hours, add 5 mL of 0.6 M NaOH solution, adjust the pH to 6.8 with 1 M HCl or 1 M NaOH, and then add freshly prepared intestinal exogenous enzyme suspension (protease: amylase: lipase = 3:1:1), seal with parafilm, and incubate in a 39°C constant temperature shaker for 18 hours (150 r / min).

[0122] After the reaction, the degradation rates of tylosin and erythromycin in the Chinese herbal medicine were 50.43% and 60.16%, respectively.

[0123] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Application of zearalenone hydrolase ZHRnZ in the degradation of zearalenone and its derivatives.

2. The use according to claim 1, characterized in that: The amino acid sequence of the corn zearalenone hydrolase ZHRnZ is as shown in SEQ ID NO.

1.

3. The use according to claim 1, characterized in that: The nucleotide sequence of the corn zearalenone hydrolase ZHRnZ is: the sequence shown in SEQ ID NO.

2.

4. A recombinant expression vector or recombinant strain comprising the zearalenone hydrolase ZHRnZ gene according to claim 3.

5. The recombinant expression vector or recombinant strain according to claim 4, characterized in that: The recombinant expression vector is pET-28a-ZHRnZ, wherein the zearalenone hydrolase ZHRnZ gene is inserted between restriction enzyme sites on the plasmid pET-28a to obtain the recombinant expression vector pET-28a-ZHRnZ; The recombinant strain includes at least one of Escherichia coli, yeast, Aspergillus, Bacillus or Lactobacillus.

6. A method for preparing the zearalenone hydrolase ZHRnZ according to any one of claims 1 to 3, characterized in that: The method comprises the following steps: (1) transforming a host cell with the recombinant expression vector encoding the zearalenone hydrolase ZHRnZ gene according to claim 4 to obtain a recombinant strain; (2) culturing the recombinant strain and inducing the expression of the zearalenone hydrolase ZHRnZ protein; (3) Isolate and purify zearalenone hydrolase ZHRnZ.

7. Use of the zearalenone hydrolase ZHRnZ according to any one of claims 1 to 3 in the degradation of macrolide antibiotics.

8. Use of the zearalenone hydrolase ZHRnZ according to any one of claims 1 to 3 in degrading at least one of zearalenone, α-zearalenol, β-zearalenol, zearalanol, erythromycin or tylosin, wherein the enzyme can completely degrade 10 μg / mL of zearalenone and its derivatives within 20 minutes.

9. An additive, characterized in that Comprising zearalenone hydrolase ZHRnZ and a physiologically acceptable carrier thereof; The content of the zearalenone hydrolase ZHRnZ is 1%-10%; The amino acid sequence of the corn zearalenone hydrolase ZHRnZ is as shown in SEQ ID NO.1; The physiologically acceptable carrier is selected from one or more of wheat, rice, oat bran, rice bran, sucrose, starch, cornmeal, maltodextrin, cyclodextrin, bentonite, montmorillonite, oligosaccharide or yeast cell wall.

10. Use of the additive according to claim 9 in the biodegradation of zearalenone and its derivatives and macrolide antibiotics; The zearalenone hydrolase ZHRnZ can degrade one or more of zearalenone, α-zearalenol, β-zearalenol, zearalenol, erythromycin and tylosin.

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