Zearalenone degrading enzyme as well as preparation method and application thereof

Abstract

The invention provides a zearalenone degrading enzyme as well as a preparation method and application thereof, and belongs to the technical field of enzyme engineering, and the zearalenone degrading enzyme comprises a nucleotide sequence as shown in SEQ ID NO.1. The zearalenone degrading enzyme provided by the invention can degrade zearalenone and derivatives thereof when the pH is acidic, neutral and alkaline.

Description

Technical Field

[0001] The present invention relates to the technical field of enzyme engineering, and in particular to a zearalenone-degrading enzyme and a preparation method and application thereof. Background Art

[0002] Zearalenone (ZEN) is a secondary metabolite of Fusarium and a non-steroidal estrogenic mycotoxin. It was first discovered in moldy corn in 1962 and is also found in many higher plants such as wheat, soybeans, cotton, etc., and acts as a hormone in plants to regulate plant growth. For animals, ZEN has estrogenic effects, mainly acting on the reproductive system, which can cause hyperestrogenism in livestock, poultry and experimental mice. Pregnant animals (including humans) who eat foods containing ZEN may suffer from miscarriage, stillbirth and malformation. Eating various pasta made with excessive ZEN can also cause central nervous system poisoning symptoms such as nausea, chills, headaches, depression and ataxia. Therefore, it is of great significance to find effective ways to reduce ZEN to improve food security.

[0003] The main methods for degrading mycotoxins are physical, chemical and biological. The physical method is unstable and easy to desorb. It also has the phenomenon of non-specific adsorption of nutrients such as vitamins, mineral elements and amino acids. The chemical method has high processing costs, and chemical residues and the introduction of new harmful components affect the safety of feed. Biological methods include microbial methods and enzyme methods, which have good degradation efficiency and high safety and have received special attention. Since microorganisms contain harmful components such as virulence factors, and the essence of microbial detoxification is to degrade mycotoxins through enzymes produced by microorganisms, enzymatic detoxification is considered to be the best method for mycotoxin detoxification.

[0004] Enzymatic detoxification of zearalenone requires the destruction of the original structure of zearalenone or the formation of low-toxic substances through biological metabolic processes. During the metabolic process, zearalenone will also produce a variety of derivatives, such as α-zearalanol (α-ZAL), β-zearalanol (β-ZAL), and zearalanone (ZAN). These derivatives still have estrogen toxicity.

[0005] At present, there are some biological enzymes that can degrade zearalenone, such as the zearalenone-degrading enzyme ZHD101 isolated from Clonostachys rosea IFO7063, and the peroxidase POD obtained by separation and purification that can be used to degrade zearalenone in beer. Although the above enzymes can be used to degrade zearalenone, the activity of the above enzymes in removing other zearalenone derivatives produced in food processing is relatively weak, and its degradation must be carried out under alkaline conditions. The above factors limit the application of the above enzymes in the food and feed industry.

[0006] Therefore, it is necessary to provide a zearalenone-degrading enzyme and a preparation method and application thereof to solve the problems existing in the above-mentioned prior art. Summary of the invention

[0007] In view of this, the present invention provides a zearalenone degrading enzyme and a preparation method and application thereof, which can degrade zearalenone and its derivatives at acidic, neutral and alkaline pH.

[0008] To achieve the above object, the present invention provides a corn zearalenone degrading enzyme, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0009] Optionally, its amino acid sequence is shown in SEQ ID NO.2.

[0010] Optionally, the use conditions of the zearalenone degrading enzyme include pH 2-8 and temperature 20-40°C.

[0011] Optionally, the use conditions of the zearalenone degrading enzyme include pH 7 and temperature 30°C.

[0012] In order to achieve the above object, the present invention also provides a zearalenone degrading enzyme and a preparation method and application thereof, comprising the following steps: S1. Synthesize the gene encoding the zearalenone-degrading enzyme of zearalenone and connect it to the secretory yeast expression vector; transform the competent cells of Pichia pastoris GS115, and perform PCR verification and sequencing to determine whether the correct recombinant strain is obtained; S2. Cultivating the recombinant strain to induce the expression of zearalenone-degrading enzyme.

[0013] Optionally, the culture of the recombinant strain includes the following steps: streaking the recombinant strain on YPD agar medium for inverted culture, then picking a single colony for culture in YPD liquid culture medium; transferring the bacterial solution to BMGY medium for continued culture, centrifuging the cultured bacterial solution at low temperature, taking the bacteria, resuspending them with pre-cooled distilled water without sterilization, centrifuging them at low temperature again, taking the bacteria, resuspending them with pre-cooled distilled water without sterilization, washing them twice, and then inoculating them in BMMY medium, and adding methanol for induction culture.

[0014] Optionally, the inverted culture condition is 30°C for 48h; when the bacterial solution is transferred to the BMGY medium, the weight ratio of the bacterial solution to the medium is 1:10, and the culture condition is 30°C, 220 rpm for 24h; the low-temperature centrifugation condition is 4°C, 5000 rpm for 5min; inoculated in the BMMY medium until the OD 600 =1.0, the culture conditions were 24°C, 220 rpm for 96 h, and the methanol concentration was 0.5%.

[0015] In order to achieve the above object, the present invention also provides a zearalenone-degrading enzyme for use in the detoxification of fungal toxins.

[0016] Optionally, the fungal toxin includes one or a combination of two or more of zearalenone, α-zearalanol, β-zearalanol and zearalanone.

[0017] Optionally, the zearalenone degrading enzyme is used as a feed additive to detoxify fungal toxins.

[0018] The above technical solution of the present invention includes at least the following beneficial effects: The zearalenone-degrading enzyme provided by the present invention is a new type of degradation enzyme, which has a high degradation rate for zearalenone and its derivatives, and can achieve a high degradation rate at neutral, acidic and alkaline pH, thereby expanding the application scenarios of the zearalenone-degrading enzyme and realizing efficient detoxification in food and feed. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The chromatogram of zearalenone in Example 2 of the present invention is shown below; Figure 2 The chromatogram of α-zearalanol in Example 3 of the present invention; Figure 3 The chromatogram of β-zearalanol in Example 4 of the present invention; Figure 4 The chromatogram of zeaxanthin in Example 5 of the present invention is shown below; Figure 5 The chromatogram of zearalenone in Example 6 of the present invention is shown below; Figure 6 This is a graph showing the effect of different temperatures on the activity of zearalenone-degrading enzyme in Test Example 1 of the present invention; Figure 7 This is a graph showing the effect of different pH values ​​on the activity of the zearalenone-degrading enzyme in Test Example 2 of the present invention; Figure 8 This is a graph showing the degradation rate of zearalenone and its derivatives by the zearalenone degrading enzyme in Examples 2 to 5 of the present invention. DETAILED DESCRIPTION

[0020] To make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the following will be combined with the appended drawings of the embodiments of the present invention. Figures 1 to 7 , the technical scheme of the embodiment of the present invention is clearly and completely described. Obviously, the described embodiment is a part of the embodiment of the present invention, not all of the embodiments. Based on the described embodiment of the present invention, all other embodiments obtained by ordinary technicians in this field belong to the scope of protection of the present invention.

[0021] The experimental reagents used in the examples of the present invention are introduced as follows: zearalenone, α-zearalanol, β-zearalanol and zearalanone standard products were purchased from Anolon; other reagents were domestic analytical grade.

[0022] Example 1 Preparation of Zearalenone-degrading Enzyme 1. Construction of strain expressing zearalenone-degrading enzyme 1.1 Construction of Zearalenone Degrading Enzyme Expression Vector The zearalenone degrading enzyme encoding gene was synthesized by Anhui General Biosystem Co., Ltd. The nucleotide sequence of the zearalenone degrading enzyme encoding gene is shown in SEQ ID NO.1 and connected to pPICZalphaA. The zearalenone degrading enzyme encoding gene was codon optimized to make it more conducive to expression in Pichia pastoris.

[0023] 1.2 Construction of strain expressing zearalenone-degrading enzyme The competent cells of Pichia pastoris GS115 were transformed, and PCR verification and sequencing were performed to determine that the correct recombinant strain was obtained. The amino acid sequence of the zearalenone degrading enzyme was shown in SEQ ID NO.2.

[0024] 2. Expression of Zearalenone-degrading Enzyme in Pichia pastoris The stored recombinant strain was streaked on YPD agar medium and inverted for 48 h at 30℃. A single colony was picked and placed in 10 ml YPD liquid medium, and cultured overnight at 30℃ 220 rpm to obtain a bacterial solution; the bacterial solution was transferred to BMGY medium at a ratio of 1:10 and cultured at 30℃ 220 rpm for 24 h; the above cultured bacterial solution was centrifuged at 5000 rpm and 4℃ for 5 min. The supernatant was discarded and the bacteria were resuspended in pre-cooled distilled water without sterilization. Centrifuge at 4℃, 5000 rpm, and 4℃ for 5 min, and repeated once. After washing twice, all were inoculated in BMMY medium to OD600=1.0, and cultured in a 250 mL baffled shake flask at 24℃ 220 rpm, and 0.5% methanol was added daily for induction culture for 96 h.

[0025] Example 2 Application of Zearalenone-degrading Enzyme in Degradation of Zearalenone (ZEN) Zearalenone was dissolved in acetonitrile to prepare a 100 μg / mL mother solution. The experiment was carried out in the following 1000 μL reaction system: 980 μL phosphate buffer (0.1M, pH 7.0), 10 μL zearalenone degrading enzyme protein (final enzyme activity 1U / mL), 10 μL zearalenone solution. The system without zearalenone degrading enzyme was used as a control. After the reaction was carried out at 30°C for 24 hours, 3 mL of methanol was added to terminate the reaction. Liquid chromatography tandem mass spectrometry was used to detect whether the zearalenone degrading enzyme had degradation activity on zearalenone based on the change in zearalenone concentration.

[0026] The chromatographic conditions for the detection of zearalenone by liquid chromatography tandem mass spectrometry are as follows: Waters liquid chromatography-triple quadrupole mass spectrometer I-CLASS PLUS / XEVO TQ-XS, chromatographic column ACQUITY UPLC® BEH C18 (1.7 μm, 2.1×50 mm), mobile phase A: acetonitrile, B: 0.1% formic acid water; flow rate: 0.3 mL / min; 0-1 min mobile phase A 10%, 1-3 min mobile phase A increased from 10% to 90% and maintained until the 5th minute, 5-7 min mobile phase A decreased from 90% to 10%. Injection volume: 5 μL; negative ion mode, the chromatogram obtained is Figure 1 .

[0027] Under the above chromatographic conditions, Figure 1 As shown, the control group sample had a strong absorption peak at retention time RT = 2.97min, while a smaller target peak was detected in the group with the addition of zearalenone degrading enzyme. According to the calculation of the absorption peak area, 80% of the zearalenone molecules had been degraded, indicating that the recombinant zearalenone degrading enzyme has the activity of degrading zearalenone.

[0028] Example 3 Application of Zearalenone-degrading Enzyme in Degradation of α-Zealanol (α-ZAL) α-Zea mays alcohol was dissolved in acetonitrile to prepare a 100 μg / mL mother solution, and the experiment was carried out according to the following 1000 μL reaction system: 980 μL phosphate buffer (0.1M, pH 7.0), 10 μL zearalenone degrading enzyme protein (final enzyme activity 1U / mL), 10 μL α-Zea mays alcohol solution. The system without zearalenone degrading enzyme was used as a control. After the reaction was carried out at 30°C for 24 hours, 3 mL of methanol was added to terminate the reaction. Liquid chromatography tandem mass spectrometry was used to detect whether the zearalenone degrading enzyme had degradation activity on α-Zea mays alcohol according to the change in α-Zea mays alcohol concentration.

[0029] The chromatographic conditions for the detection of α-zearalanol by liquid chromatography tandem mass spectrometry are as follows: Waters liquid chromatography-triple quadrupole mass spectrometer I-CLASS PLUS / XEVO TQ-XS, chromatographic column ACQUITY UPLC® BEH C18 (1.7 μm, 2.1×50 mm), mobile phase A: acetonitrile, B: 0.1% formic acid water; flow rate: 0.3 mL / min; 0-1 min mobile phase A 10%, 1-3 min mobile phase A increased from 10% to 90% and maintained until the 5th minute, 5-7 min mobile phase A decreased from 90% to 10%. Injection volume: 5 μL; negative ion mode, the chromatogram is as follows Figure 2 .

[0030] Under the above chromatographic conditions, Figure 2 As shown, the control group sample had a strong absorption peak at the retention time RT = 2.94min, while a smaller target peak was detected in the group added with zearalenone-degrading enzyme. According to the calculation of the absorption peak area, 53% of the α-zearalanol molecules had been degraded, indicating that the recombinant zearalanone-degrading enzyme has the activity of degrading α-zearalanol.

[0031] Example 4 Application of Zearalenone-degrading Enzyme in Degradation of β-Zealanol (β-ZAL) β-Zea mays was dissolved in acetonitrile to prepare a 100 μg / mL mother solution. The experiment was carried out in the following 1000 μL reaction system: 980 μL phosphate buffer (0.1M, pH 7.0), 10 μL zearalenone degrading enzyme protein (final enzyme activity 1U / mL), 10 μL β-Zea mays solution. The system without zearalenone degrading enzyme was used as a control. After the reaction was carried out at 30°C for 24 hours, 3 mL of methanol was added to terminate the reaction. Liquid chromatography tandem mass spectrometry was used to detect whether the zearalenone degrading enzyme had degradation activity on β-Zea mays according to the change in β-Zea mays concentration.

[0032] The chromatographic conditions for the detection of β-zearalanol by liquid chromatography tandem mass spectrometry are as follows: Waters liquid chromatography-triple quadrupole mass spectrometer I-CLASS PLUS / XEVO TQ-XS, chromatographic column ACQUITY UPLC® BEH C18 (1.7 μm, 2.1×50 mm), mobile phase A: acetonitrile, B: 0.1% formic acid water; flow rate: 0.3 mL / min; 0-1 min mobile phase A 10%, 1-3 min mobile phase A increased from 10% to 90% and maintained until the 5th minute, 5-7 min mobile phase A decreased from 90% to 10%. Injection volume: 5 μL; negative ion mode, the chromatogram is as follows Figure 3 .

[0033] Under the above chromatographic conditions, Figure 3 As shown, the control group sample had a strong absorption peak at the retention time RT = 2.95min, while a smaller target peak was detected in the group added with zearalenone-degrading enzyme. According to the calculation of the absorption peak area, 47% of the β-zearalanol molecules had been degraded, indicating that the recombinant zearalanone-degrading enzyme has the activity of degrading β-zearalanol.

[0034] Example 5 Application of Zearalenone-degrading Enzyme in Degradation of Zearalenone (ZAN) Zearalenone was dissolved in acetonitrile to prepare a 100 μg / mL mother solution, and the experiment was carried out according to the following 1000 μL reaction system: 980 μL phosphate buffer (0.1M, pH 7.0), 10 μL zearalenone degrading enzyme protein (final enzyme activity 1U / mL), 10 μL zearalenone solution. The system without zearalenone degrading enzyme was used as a control. After the reaction was carried out at 30°C for 24 hours, 3 mL of methanol was added to terminate the reaction. Liquid chromatography tandem mass spectrometry was used to detect whether the zearalenone degrading enzyme had degradation activity on zearalenone based on the change in zearalenone concentration.

[0035] The chromatographic conditions for the detection of zearalanone by liquid chromatography tandem mass spectrometry are as follows: Waters liquid chromatography-triple quadrupole mass spectrometer I-CLASS PLUS / XEVO TQ-XS, chromatographic column ACQUITY UPLC® BEH C18 (1.7 μm, 2.1×50 mm), mobile phase A: acetonitrile, B: 0.1% formic acid water; flow rate: 0.3 mL / min; 0-1 min mobile phase A 10%, 1-3 min mobile phase A increased from 10% to 90% and maintained until the 5th minute, 5-7 min mobile phase A decreased from 90% to 10%. Injection volume: 5 μL; negative ion mode, the chromatogram obtained is Figure 4 .

[0036] Under the above chromatographic conditions, Figure 4 As shown, the control group sample had a strong absorption peak at retention time RT = 2.97min, while a smaller target peak was detected in the group added with zearalenone-degrading enzyme. According to the calculation of the absorption peak area, 88% of the zearalenone molecules had been degraded, indicating that the recombinant zearalenone-degrading enzyme has the activity of degrading zearalenone.

[0037] Example 6 Zearalenone-degrading enzyme as a feed additive to treat zearalenone in corncob meal.

[0038] The zearalenone-degrading enzyme was dissolved in phosphate buffer (20 mM, pH 7.0) at a concentration of 10 U / mL to obtain a feed additive, which was stored at 4°C or room temperature.

[0039] Naturally moldy corncob powder was put into tissue culture bottles, 5 g of corncob powder in each bottle, and an appropriate amount of water was added to fully dissolve the corncob powder. 1 mL of additive was added as the treatment group, and 1 mL of sterile water was added to the control group. The mixture was shaken well and cultured at a constant temperature of 30°C for 24 h. Zearalenone in the corncob powder was detected by liquid chromatography-tandem mass spectrometry.

[0040] The chromatographic conditions for the detection of β-zearalanol by liquid chromatography tandem mass spectrometry were as follows: Waters liquid chromatography-triple quadrupole mass spectrometer I-CLASS PLUS / XEVO TQ-XS, chromatographic column ACQUITY UPLC® BEH C18 (1.7 μm, 2.1×50 mm), mobile phase A: acetonitrile, B: 0.1% formic acid water; flow rate: 0.3 mL / min; mobile phase A 10% from 0 to 1 min, mobile phase A increased from 10% to 90% from 1 to 3 min and maintained until the 5th min, and mobile phase A decreased from 90% to 10% from 5 to 7 min. Injection volume: 5 μL; negative ion mode.

[0041] The degradation rate of zearalenone was determined to be 75.86%. Figure 5 shown.

[0042] Test Example 1 Effect of reaction temperature on the activity of zearalenone-degrading enzyme in degrading zearalenone Compared with Example 2, the only difference is that the reaction temperatures are 20°C, 40°C and 60°C respectively, and the other reaction systems and reaction times are the same. Figure 6 .

[0043] Test Example 2 Effect of pH on the activity of zearalenone-degrading enzyme in degrading zearalenone Compared with Example 2, the only difference is the pH, which is 0.1M glycine-HCl, pH 2; 0.1MNa2HPO4-sodium citrate buffer, pH 3-7; 0.1M Tris-HCl buffer, pH 8-9; 0.1M glycine-NaOH buffer, pH 10. The other reaction systems and reaction time conditions are the same. After testing, Figure 7 .

[0044] Depend on Figures 1 to 4 It can be seen from Examples 2 to 5 that the zearalenone-degrading enzyme can degrade zearalenone, α-zearalanol, β-zearalanol and zearalanone.

[0045] Depend on Figure 8It can be seen that the degradation rates of zearalenone, α-zearalanol, β-zearalanol and zearalanone by zearalenone-degrading enzyme are different, and the degradation rate of zearalanone and zearalanone is the highest, reaching 88%, and the degradation rate of β-zearalanol is the lowest, reaching 47%.

[0046] The temperature is controlled in the test case. Figure 5 It can be seen that temperature affects the activity of zearalenone-degrading enzyme. At 30°C, the degradation rate of zearalenone-degrading enzyme is the highest. Above 30°C, the degradation rate of zearalenone-degrading enzyme gradually decreases.

[0047] The pH value was controlled in the test case. Figure 7 It can be seen that pH affects the activity of zearalenone degrading enzyme. When the pH is 7, the degradation rate of zearalenone degrading enzyme is the highest. When the pH is acidic, the degradation rate of zearalenone degrading enzyme is reduced, but it can be maintained at more than 70%. When the pH is alkaline, the degradation rate of zearalenone degrading enzyme drops suddenly. Therefore, the zearalenone degrading enzyme obtained by the present invention has a high degradation rate when the pH is acidic and neutral, and has few restrictions on application conditions.

[0048] The SEQ ID NO.1 of this application is as follows: <160> 1 <211> 1161 <212> DNA <213> Aspergillus sclerotiniger <221> Zearalenone-degrading enzyme gene <400> 1 The SEQ ID NO.2 of the present application is as follows: <211> 387 <212> PRT <213> Aspergillus sclerotiniger <221> zearalenone-degrading enzyme <400> 2 mrlalvplff gatavasrly aasyagtvtt lslsqsrgqy eletiaqste cginpswlml 61 dsehrvlycl deavdlangt ltslaigang tltrivqret iagpvmsqfy sapglpdrne 121 gsavttysld pitgvfnrsq tftytlaqpg pnaarqdaph shgvvvdptg qfvlvpdlga 181 dfvrifrvnp stgllepqdp lvaapgsgpr hgvfwtpkda kvgqhtetif ylvselsngl 241 tgyrvsypng tiafsefytt nsyggavapn askvaeihls pqnnrivlsn rgdntfgvnn 301 dsmavfgcad atgniatnit fnglypaygs yprqydlsak nemiaealqe sdavvvtgwn 361 gttgkpgpfl arkslvgeiv avvwdsl The above are preferred embodiments 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 principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A zearalenone-degrading enzyme, characterized in that: Its nucleotide sequence is shown in SEQ ID NO.

1.

2. The zearalenone degrading enzyme according to claim 1, characterized in that Its amino acid sequence is shown in SEQ ID NO.

2.

3. The zearalenone-degrading enzyme according to claim 1, characterized in that The use conditions of the zearalenone degrading enzyme include pH 2-8 and temperature 20-40°C.

4. The zearalenone-degrading enzyme according to claim 3, characterized in that The use conditions of the zearalenone degrading enzyme include pH 7 and temperature 30°C.

5. A method for preparing the zearalenone degrading enzyme according to any one of claims 1 to 4, characterized in that: The following steps are involved: S1. Synthesize the gene encoding the zearalenone-degrading enzyme of zearalenone and connect it to the secretory yeast expression vector; transform the competent cells of Pichia pastoris GS115, and perform PCR verification and sequencing to determine whether the correct recombinant strain is obtained; S2. Cultivating the recombinant strain to induce the expression of zearalenone-degrading enzyme.

6. The method for preparing the zearalenone-degrading enzyme according to claim 5, characterized in that: The recombinant strain culture comprises the following steps: streaking the recombinant strain on a YPD agar medium for inverted culture, picking a single colony for culture in a YPD liquid medium; transferring the bacterial solution to a BMGY medium for continued culture, centrifuging the cultured bacterial solution at low temperature, taking the bacterial bodies, resuspending them in precooled distilled water without sterilization, centrifuging at low temperature again, taking the bacterial bodies, resuspending them in precooled distilled water without sterilization, washing them twice, and then inoculating them in a BMMY medium, and adding methanol for induction culture.

7. The method for preparing the zearalenone-degrading enzyme according to claim 6, characterized in that: The inverted culture condition is 30°C for 48h; when the bacterial solution is transferred to the BMGY medium, the weight ratio of the bacterial solution to the medium is 1:10, and the culture condition is 30°C, 220 rpm for 24h; the low-temperature centrifugation condition is 4°C, 5000 rpm for 5min; inoculated in the BMMY medium until the OD 600 =1.0, the culture conditions were 24°C, 220 rpm for 96 h, and the methanol concentration was 0.5%.

8. A zearalenone-degrading enzyme according to any one of claims 1 to 4 used in the detoxification of fungal toxins.

9. The zearalenone degrading enzyme according to claim 8 is used for the detoxification of fungal toxins, characterized in that: The fungal toxins include one or a combination of two or more of zearalenone, α-zearalanol, β-zearalanol and zearalanone.

10. The zearalenone degrading enzyme according to claim 8 is used for the detoxification of fungal toxins, characterized in that: The zearalenone degrading enzyme is used as a feed additive to detoxify fungal toxins.

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