Coding gene of aflatoxin B1 and zearalenone degrading enzyme and application thereof

Abstract

The invention belongs to the technical field of enzyme engineering, and relates to degradation of aflatoxin B1 and zearalenone, in particular to a coding gene of an aflatoxin B1 and zearalenone degrading enzyme and application of the coding gene. The coding gene has a similarity of 90% or more with a nucleotide sequence shown as SEQ ID No.2. The coding gene has a nucleotide sequence shown as SEQ ID No.2. The amino acid coded by the gene has 70% or more similarity with the amino acid sequence shown in SEQ ID No.1. The aflatoxin B1 and zearalenone degrading enzyme disclosed by the invention is used for degrading AFB1 and ZEN, the optimum temperature for degrading the AFB1 is 40 DEG C, and the pH is 7.0; the optimal temperature for ZEN is 60 DEG C, and the pH value is 8.0; the degradation rate of the enzyme to AFB1 is 98.82%, the time is prolonged to 36 h, and the highest degradation rate is 99.87%; the degradation rate of ZEN is 96.1%, the time is prolonged to 36 h, and the highest degradation rate is 99.33%.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of enzyme engineering, and relates to degradation of mycotoxins, in particular to a coding gene of a aflatoxin B1 and zearalenone degrading enzyme and application thereof. BACKGROUND

[0002] In the past few decades, mycotoxin contamination in food and feed has become a major global problem. According to the data of the Food and Agriculture Organization of the United Nations (FAO) in 1985, more than 25% of crops worldwide were contaminated by mycotoxins. Further studies have shown that the previous estimate of mycotoxin contamination by the Food and Agriculture Organization was underestimated. Data from about 500,000 analyses conducted by the European Food Safety Authority (EFSA) and a large global mycotoxin survey showed that the contamination rate was actually as high as 60-80%. Eight assessments of feed and raw materials worldwide showed that 72% of samples were contaminated with at least one mycotoxin, and 38% of samples contained multiple mycotoxins. In general, co-occurring mycotoxin contamination can cause more serious health problems than single mycotoxin contamination.

[0003] Aflatoxin B1 (AFB1) and zearalenone (ZEN) often coexist in corn, wheat and rice and other cereals, posing a major threat to human and animal health. AFB1 is the most toxic mycotoxin, with hepatotoxicity, teratogenicity and carcinogenicity, mainly produced by Aspergillus flavus, Aspergillus parasiticus and Aspergillus terreus. It is associated with cancer, immunosuppression and hepatotoxicity, and is widely present in various foods. ZEN is a mycotoxin produced by Fusarium, which contaminates food and cereals worldwide, especially corn. It is one of the most widely distributed mycotoxins, mainly affecting the reproductive system, causing pregnant animals to abort and stillbirth. Exposure to various mycotoxins, including those from the same or different species, can cause combined toxicity due to interaction. Depending on the combination of mycotoxins, joint toxicity can be synergistic, additive or antagonistic. For example, AFs combined with different AFs or other carcinogenic mycotoxins can cause joint toxicity. The combination of AFB1 and ZEN can cause synergistic cytotoxicity. In addition, mycotoxins cause huge economic losses to the food and livestock sectors every year.

[0004] Many methods, including biological, chemical and physical methods, have been used to detoxify mycotoxins from grains and feed. But the biological detoxification method is poor in specificity, unstable in detoxification effect, and the chemical detoxification has the problems of nutrient loss, difficult implementation, easy secondary pollution, etc. In recent years, biological detoxification has become a research hotspot due to its high efficiency, short cycle, high safety, strong specificity and environmental friendliness. More and more researchers have begun to focus on biological methods based on microorganisms or their enzymes. Enzymatic detoxification can avoid the potential toxicity of microorganisms and the harm caused by changes in raw materials and sensory properties. Several enzymes isolated from many microorganisms, including manganese peroxidase (MnP), Pseudomonas AFB1-degrading enzyme (PADE) and laccase, have shown biological transformation activity on aflatoxins. In addition, microbially derived enzymes such as Ase, ZEN hydrolytic enzyme (ZHD) and ZEN hydrolytic enzyme 101 (Zhd101) have been reported to be able to degrade ZEN. Most of the reported degrading enzymes can only produce degradation effect on one kind of mycotoxin, but considering the diversity of mycotoxin pollution, single-specific toxin degradation has been unable to meet the actual demand, and a multifunctional enzyme capable of simultaneously degrading multiple mycotoxins is urgently needed. Publication No. CN 113563481 A discloses that two enzymes, zearalenone hydrolytic enzyme ZHD101.1 and manganese peroxidase PhcMnp, are fused to obtain a fusion enzyme capable of simultaneously degrading ZEN and AFB1, with a degradation rate of 46.46% for AFB1 and 82.05% for ZEN. Publication No. CN 113699126 A discloses the application of a dye decolorizing peroxidase StDyP capable of simultaneously degrading AFB1 and ZEN, with a degradation rate of 21.03% for AFB1 and 45.37% for ZEN. Therefore, it is of important and urgent significance to isolate, identify and develop new enzymes capable of simultaneously degrading AFB1 and ZEN. SUMMARY

[0005] To solve the above technical problems, the present application provides an aflatoxin B1 and zearalenone degrading enzyme coding gene and its application.

[0006] The technical solution of the present application is as follows: An aflatoxin B1 and zearalenone degrading enzyme coding gene has a similarity of more than 90% with the nucleotide sequence shown in SEQ ID No. 2. The nucleotide sequence with a similarity of more than 90% with the nucleotide sequence of the present application can express the amino acid sequence of the present application, for example, SEQ ID No. 3, which is the nucleotide sequence after codon optimization of Escherichia coli, and the sequence has a similarity of more than 90% with the gene sequence shown in SEQ ID No. 2.

[0007] Preferably, the nucleotide sequence of the above-mentioned gene is as shown in SEQ ID No. 2 or SEQ ID No. 3.

[0008] An aflatoxin B1 and zearalenone degrading enzyme having more than 70% similarity with the amino acid sequence shown in SEQ ID No. 1. An amino acid sequence having more than 70% similarity with the amino acid sequence of the present application can retain the degradation function of the aflatoxin B1 and zearalenone degrading enzyme of the present application.

[0009] Further, the aflatoxin B1 and zearalenone degrading enzyme described above has an amino acid sequence as shown in SEQ ID No. 1.

[0010] A recombinant plasmid containing the above-mentioned gene.

[0011] An engineered bacterium containing the above-mentioned recombinant plasmid.

[0012] The engineered bacterium is Escherichia coli.

[0013] The aflatoxin B1 and zearalenone degrading enzyme can be applied in the field of detoxification of aflatoxin B1 and zearalenone.

[0014] The temperature for the aflatoxin B1 and zearalenone degrading enzyme to degrade aflatoxin B1 and zearalenone is 20-80℃, and the pH is 5.0-10.0.

[0015] The optimal temperature for the aflatoxin B1 and zearalenone degrading enzyme to degrade aflatoxin B1 is 40℃, and the pH is 7.0.

[0016] The aflatoxin B1 and zearalenone degrading enzyme can maintain a degradation rate of more than 75% of aflatoxin B1 in the temperature range of 40-80℃.

[0017] The optimal temperature for the aflatoxin B1 and zearalenone degrading enzyme to degrade zearalenone is 60℃, and the pH is 8.0.

[0018] The aflatoxin B1 and zearalenone degrading enzyme can maintain a degradation rate of more than 75% of aflatoxin B1 in the temperature range of 50-80℃.

[0019] The aflatoxin B1 and zearalenone degrading enzyme is an enzyme preparation.

[0020] Preferably, the addition amount of the aflatoxin B1 and zearalenone degrading enzyme for aflatoxin B1 contamination is 20 μg / mL, and the addition amount for zearalenone contamination is 10 μg / mL.

[0021] A biological reagent for simultaneously degrading aflatoxin B1 and zearalenone, the biological reagent being selected from any one or a combination of the following: (1) a gene encoding aflatoxin B1 and zearalenone degrading enzyme as described above; (2) aflatoxin B1 and zearalenone degrading enzyme as described above; (3) the recombinant plasmid as described above; (4) the engineering bacteria as described above; (5) the enzyme preparation as described above.

[0022] The effective component in the biological reagent is aflatoxin B1 and zearalenone degrading enzyme.

[0023] The aflatoxin B1 and zearalenone degrading enzyme can simultaneously degrade AFB1 and ZEN, the degradation rate of AFB1 reaching 92.8%, and the degradation rate of ZEN reaching 93.9%.

[0024] The present application has the following beneficial effects: 1. The present application discloses that the aflatoxin B1 and zearalenone degrading enzyme is used for degrading AFB1 and ZEN, the optimal temperature for degrading AFB1 being 40 DEG C, and the pH being 7.0; the optimal temperature for degrading ZEN being 60 DEG C, and the pH being 8.0; the degradation rate of the enzyme to AFB1 being 98.82%, the time being extended to 36h, and the highest degradability being 99.87%; the degradation rate of the enzyme to ZEN being 96.1%, the time being extended to 36h, and the highest degradability being 99.33%.

[0025] 2. Food and feed are contaminated by various mycotoxins, which has become a major global problem, and the combined contamination of mycotoxins can cause more serious health problems, so it is of great and urgent significance to identify and develop new enzymes capable of simultaneously degrading AFB1 and ZEN in food and feed. The mycotoxin degrading enzyme disclosed in the present application can simultaneously degrade AFB1 and ZEN, the degradation rate of AFB1 reaching 92.8%, and the degradation rate of ZEN reaching 93.4% (see Figure 11 ). BRIEF DESCRIPTION OF DRAWINGS

[0026] In order to more clearly illustrate the technical solutions of the embodiments of the present application or the prior art, the following will briefly introduce the drawings needed to be used in the embodiments or prior art description. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without any creative effort.

[0027] Figure 1 The plasmid map of pET-28a(+).

[0028] Figure 2 Figure 6 is a map of pET-28a(+) -degrading enzyme recombinant plasmid.

[0029] Figure 3 Figure 7 is a PCR electrophoresis map of bacterial liquid.

[0030] Figure 4 Figure 8 is a SDS-PAGE electrophoresis map of different components; wherein M: protein standard Marker; 1: uninduced; 2: after induction; 3: after crushing; 4: precipitation; 5: supernatant.

[0031] Figure 5 Figure 9 is a SDS-PAGE electrophoresis map of purified degrading enzyme.

[0032] Figure 6 Figure 10 is a degradation of AFB1 and ZEN by degrading enzyme under different pH conditions.

[0033] Figure 7 Figure 11 is a degradation of AFB1 and ZEN by degrading enzyme under different temperature conditions.

[0034] Figure 8 Figure 12 is a degradation of AFB1 and ZEN by degrading enzyme under different metal ions.

[0035] Figure 9 Figure 13 is a degradation of AFB1 and ZEN by degrading enzyme under different reaction time.

[0036] Figure 10 Figure 14 is a simultaneous degradation of AFB1 and ZEN by degrading enzyme under different temperature.

[0037] Figure 11 Figure 15 is a simultaneous degradation of AFB1 and ZEN by degrading enzyme under different reaction time. DETAILED DESCRIPTION

[0038] The technical solutions of the present application will be described clearly and completely below in combination with the embodiments of the present application. Obviously, the described embodiments are only some of the embodiments of the present application, but not all the embodiments. Based on the embodiments in the present application, all the other embodiments obtained by those skilled in the art without creative labor are within the protection scope of the present application.

[0039] Biological material: Vector and strain: E. coli expression vector pET-28a(+), purchased from Wuhan Moli Bio-technology Co., Ltd.; E. coli DH5α, E. coli BL21(DE3), purchased from GenScript Biotech Corporation (Shanghai) Co., Ltd.

[0040] LB medium: 10 g tryptone, 5 g yeast extract powder, 10 g sodium chloride, pH 7.0, constant volume to 1 L, adjust pH = 7.0.

[0041] Experimental reagents: AFB1 and ZEN standard samples were purchased from Shanghai Yuan Ye Biotechnology Co., Ltd.; other reagents were domestic analytical pure. The nucleotide sequence of the degradation enzyme coding gene shown in SEQ ID No. 2 was synthesized by Shengong Biotechnology (Shanghai) Co., Ltd., and the codon was optimized according to the preference of E. coli BL21 (DE3). The optimized sequence is shown in SEQ ID No. 3. The restriction enzyme cutting site Sac I was introduced at the 5' end of the coding gene, and the restriction enzyme cutting site Not I was introduced at the 3' end.

[0042] The amino acid sequence of the gene is shown in SEQ ID No. 1. The gene with the nucleotide sequence shown in SEQ ID No. 3 was connected to the corresponding site of the E. coli expression vector pET-28a (+) Figure 1 ), to obtain the recombinant plasmid pET-28a (+)-degradation enzyme (as shown in Figure 2 ). The obtained recombinant plasmid was added to the competent cells of E. coli DH5α, and after 42°C water bath heat shock for 45 seconds, it was immediately placed on ice for cooling for 2-3 min. The recombinant product was transformed, and the correct strain was extracted for plasmid extraction. The E. coli expression strain BL21 (DE3) competent cells were introduced, and after 42°C water bath heat shock for 45 seconds, they were immediately placed on ice for cooling for 3 min. 900 μL of LB medium was added, and after 37°C, 200 rpm shaking for 1 h, 5000 rpm centrifugation for 5 min, 700 μL supernatant was discarded, and the remaining medium was used to resuspend the bacterial cells. A sterile spreader was used to evenly spread on the plate containing kanamycin resistance. After 12-16 h of inverted culture in a 37°C incubator, positive clones were selected, and bacterial liquid PCR verification was performed, as shown in Figure 3 , the recombinant E. coli with successful transformation was obtained.

[0043] The PCR amplification system and amplification program are shown in Tables 1 and 2.

[0044] Table 1 PCR amplification system Table 2 PCR amplification program Example 2: Induction expression and purification of aflatoxin B1 and zearalenone degradation enzyme I. Induction expression of aflatoxin B1 and zearalenone degradation enzyme BL21(DE3) / pET-28a(+)-degradase was inoculated in 50 mL of LB medium, and cultured at 37°C with 200 rpm for 16 h, then inoculated in 100 mL of LB liquid medium at 2% inoculation amount, and cultured at 37°C with 200 rpm until OD600=0.6-0.8, then 0.5 mM of IPTG was added, and induced at 16°C for 20-24 h. II. Purification of aflatoxin B1 and zearalenone degrading enzyme The induced bacterial liquid was collected, centrifuged at 8000 rpm for 5 min at 4°C, and the supernatant was discarded; the bacterial body was resuspended with 5 mL of equilibrium buffer, and ultrasonically broken for 25 min (ultrasonic power 280 W, ultrasonic on for 4 s, ultrasonic off for 4 s) under ice bath condition, the broken sample was centrifuged at 12000 rpm for 10 min at 4°C, the supernatant was collected and filtered with a 0.45 µm water filter membrane, and the precipitate was resuspended with equilibrium buffer solution and stored.

[0045] Thereafter, the obtained supernatant was purified with a nickel ion affinity chromatography column (Ni-NTA), the column was equilibrated with 10 volumes of equilibrium buffer solution, then the sample was loaded, the flow rate was controlled at 0.25-0.5 mL / min, then the protein was eluted with a prepared 250 mM imidazole eluent, and the eluate was collected; the obtained eluate was ultrafiltered at 4°C with a 30 kDa ultrafilter tube at 4000 rpm for 30 min to remove imidazole, and the purified protein was obtained. The purification effect of the purified protein was detected by SDS-PAGE gel electrophoresis, and the results are shown in Figure 5 .

[0046] Example 3: Detection of degradation rate of purified degrading enzyme on AFB1 and ZEN Experimental method: The protein concentration of the purified protein obtained in Example 2 was determined using a Bradford protein detection kit, and stored at 4°C for standby. The degradation reaction of degrading enzyme on AFB1 was carried out in a system of 500 µL, containing 5.0 μg / mL of AFB1, 20 μg of aflatoxin B1 and zearalenone degrading enzyme protein, and 50 mM of Tris-HCl (containing 1.0 mM of metal ions, pH 7.0) buffer solution. After 24 h of incubation, 500 µL of methanol was added to terminate the reaction, the system was mixed uniformly, then filtered with a 0.22 μm organic filter membrane, and the content of AFB1 was determined by high performance liquid chromatograph.

[0047] The chromatographic conditions for detecting AFB1 by high performance liquid chromatograph are as follows: chromatographic column: Proshell 120-C18 column (150 x 4.6 mm, 4 μm); ultraviolet detector: detection wavelength 365 nm; flow rate: 1 mL / min; injection volume: 20 μL; collection time: 10 min; column temperature: 35 °C; mobile phase: methanol: acetonitrile: water = 2:2:6 (v / v / v).

[0048] The calculation method of AFB1 degradation rate is as follows: Degradation rate = (C0- C1) / C0 x 100%.

[0049] The degradation reaction of ZEN by the degradation enzyme is carried out in a system of 500 μL, containing 10 μg / mL ZEN, 10 μg degradation enzyme, 50 mM Tris-HCl (containing 1.0 mM metal ions, pH 7.0) buffer solution. After 12 h of co-incubation, 500 μL of methanol is added to terminate the reaction, the system is uniformly mixed, then filtered by using a 0.22 μm organic filter membrane, and the content of ZEN is determined by using a high performance liquid chromatograph.

[0050] The chromatographic conditions for detecting AFB1 by high performance liquid chromatograph are as follows: chromatographic column: Proshell 120-C18 column (150 x 4.6 mm, 4 μm); fluorescence detector: excitation wavelength 235 nm, emission wavelength 470 nm; flow rate: 1 mL / min; injection volume: 20 μL; collection time: 5 min; column temperature: 35 °C; mobile phase: methanol: water = 8:2 (v / v). The calculation method of ZEN degradation rate is as follows: Degradation rate = (C0- C1) / C0 x 100%.

[0051] I. Effect of pH on the degradation of AFB1 and ZEN by the degradation enzyme In order to study the effect of different pH on the degradation of AFB1 by the degradation enzyme, the reaction system in Example 2 is used, and the reaction is carried out in the range of pH 5.0-10.0 (50 mM phosphate buffer solution, pH 5.0-6.0; 50 mM Tris-HCl buffer solution, pH 7.0-8.0; 50 mM Gly-NaOH buffer solution, pH 9.0-10.0; 1 mM CuSO4, pH 5.0-10.0), and the results are shown in 2+ Figure 6 As shown in the results, the degradation rate of AFB1 by the degradation enzyme is relatively high under neutral and alkaline conditions, and the optimum pH is 7.0.

[0052] ​​​To study the effect of different pH on the degradation of ZEN by the degradation enzyme, the reaction system in Example 2 was used, and the reaction was carried out at pH 5.0-10.0 (50 mM phosphate buffer solution, pH 5.0-6.0; 50 mM Tris-HCl buffer solution, pH 7.0-8.0; 50 mM Gly-NaOH buffer solution, pH 9.0-10.0; 1 mM Cu 2+ ) range, and the results are shown in Figure 6 , under alkaline conditions, the degradation rate of ZEN by the degradation enzyme is relatively high, and the optimum pH is 8.0.

[0053] II. Effect of temperature on degradation of AFB1 and ZEN by degradation enzyme To study the degradation of AFB1 by the degradation enzyme under different temperature conditions, the reaction system in Example 2 was used, and the reaction was carried out at pH=7.0 (50 mM Tris-HCl, 1 mM Cu 2+ ) under different temperatures (20, 30, 40, 50, 60, 70, 80℃), and the results are shown in Figure 7 , the degradation rate of AFB1 is the highest at 40℃, which can reach 98.82%.

[0054] To study the degradation of ZEN by the degradation enzyme under different temperature conditions, the reaction system in Example 2 was used, and the reaction was carried out at pH=8.0 (50 mM Tris-HCl, 1 mM Cu 2+ ) under different temperatures (20, 30, 40, 50, 60, 70, 80℃), and the results are shown in Figure 7 , the degradation rate of ZEN is the highest at 60℃, which can reach 96.10%.

[0055] III. Effect of metal ions on degradation of AFB1 and ZEN by degradation enzyme To study the effect of different metal ions on the degradation of AFB1 by the degradation enzyme, different concentrations of 1 mM different cations were added to the system in Example 2 under the previously determined optimum pH=7.0 and 40℃. It can be seen from Figure 8 that the addition of copper ions can significantly improve the degradation rate of AFB1 by the degradation enzyme.

[0056] To study the effect of different metal ions on the degradation of ZEN by the degradation enzyme, different concentrations of 1 mM different cations were added to the system in Example 2 under the previously determined optimum pH=8.0 and 60℃. It can be seen from Figure 8 that the addition of copper ions, manganese ions and cobalt ions can improve the degradation rate of ZEN by the degradation enzyme, among which the addition of copper ions has the highest degradation rate of ZEN by the degradation enzyme.

[0057] IV. Effect of reaction time on degradation of AFB1 and ZEN by the degradation enzyme To study the effect of different reaction times on the degradation of AFB1 by the degradation enzyme, the reaction system of Example 2 was used: 55.0 μg / mL AFB1, 20 μg of the degradation enzyme, 50 mM Tris-HCl (containing 1.0 mM Cu 2+ , pH 7.0) buffer solution, and the reaction was carried out at 40°C for different reaction times, and the degradation rate was calculated by the formula: Figure 9 It can be seen that as the reaction time is prolonged, the degradation rate increases, and reaches 99.87% at 36 h. However, after 24 h, the degradation rate does not increase significantly, and considering economic benefits, the degradation time can be selected as 24 h.

[0058] To study the effect of different reaction times on the degradation of ZEN by the degradation enzyme, the reaction system of Example 2 was used: 10.0 μg / mL ZEN, 10 μg of the degradation enzyme, 50 mM Tris-HCl (containing 1.0 mM Cu 2+ , pH 8.0) buffer solution, and the reaction was carried out at 60°C for different reaction times, and the degradation rate was calculated by the formula: Figure 9 It can be seen that as the reaction time is prolonged, the degradation rate increases, and reaches 99.33% at 36 h. After 12 h of reaction time, the degradation rate increases slowly, and considering economic benefits, the degradation time can be selected as 12 h.

[0059] V. Effect of reaction temperature on simultaneous degradation of AFB1 and ZEN by the degradation enzyme To study the simultaneous degradation of AFB1 and ZEN by the degradation enzyme, the degradation reaction system was as follows: 5.0 μg / mL AFB1, 10.0 μg / mL ZEN, 20 μg of the degradation enzyme, 50 mM Tris-HCl (containing 1.0 mM Cu 2+ , pH 8.0) buffer solution, after 24 h of incubation, 500 μL of methanol was added to terminate the reaction, the system was mixed uniformly and then filtered using a 0.22 μm organic filter membrane, and the contents of AFB1 and ZEN were determined using a high-performance liquid chromatograph. As shown in Figure 10 As the temperature increases, the degradation rate of AFB1 and ZEN by the degradation enzyme increases, and as the temperature continues to increase, the degradation rate shows a downward trend, and at 60°C, the degradation rate of AFB1 and ZEN by the degradation enzyme is relatively high, the degradation rate of AFB1 is 91.24%, and the degradation rate of ZEN is 92.19%.

[0060] VI. Effect of different reaction times on simultaneous degradation of AFB1 and ZEN by the degradation enzyme In order to study the simultaneous degradation of AFB1 and ZEN by the degradation enzyme, the degradation reaction system is as follows: 5.0 μg / mL AFB1, 10.0 μg / mL ZEN, 20 μg degradation enzyme, 50 mM Tris-HCl (containing 1.0 mM Cu 2+ , pH 8.0) buffer solution, after incubation at 60°C for different times, 500 μL of methanol was added to terminate the reaction, the system was mixed uniformly and then filtered by using a 0.22 μm organic filter membrane, and the contents of AFB1 and ZEN were determined by using a high performance liquid chromatograph. As shown in Figure 11 , with the extension of the reaction time, the degradation rates of AFB1 and ZEN by the degradation enzyme increased. After different reaction times, the degradation rate of AFB1 was better than that of ZEN. After 27 h of reaction time, the degradation rate of AFB1 was 92.81%, and the degradation rate of ZEN was 93.93%. The above only describes the preferred embodiments of the present application and is not intended to limit the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A gene encoding an aflatoxin B1 and zearalenone degrading enzyme having more than 90% similarity to the nucleotide sequence shown in SEQ ID No.

2.

2. The gene encoding an aflatoxin B1 and a zearalenone-degrading enzyme according to claim 1, characterized in that: The nucleotide sequence of the gene is shown in SEQ ID No. 2 or SEQ ID No.

3.

3. An aflatoxin B1 and zearalenone degrading enzyme having more than 70% similarity to the amino acid sequence shown in SEQ ID No.

1.

4. The aflatoxin B1 and zearalenone degrading enzyme according to claim 3, having the amino acid sequence shown in SEQ ID No.

1.

5. A recombinant plasmid containing the gene according to claim 1 or 2.

6. An engineered bacterium containing the recombinant plasmid according to claim 5.

7. The engineered bacteria containing recombinant plasmid according to claim 6, characterized in that: The engineered bacterium is Escherichia coli.

8. An enzyme preparation containing the aflatoxin B1 and zearalenone degrading enzyme according to claim 3 or 4.

9. A biological agent for simultaneously degrading aflatoxin B1 and zearalenone, characterized by, The biological agent is selected from any one or a combination of the following: (1) the gene encoding an aflatoxin B1 and zearalenone degrading enzyme according to claim 1 or 2; (2) the aflatoxin B1 and zearalenone degrading enzyme according to claim 3 or 4; (3) the recombinant plasmid according to claim 5; (4) the engineered bacterium according to claim 6 or 7; (5) the enzyme preparation according to claim 8.

10. The biological agent for simultaneous degradation of aflatoxin B1 and zearalenone according to claim 9, characterized by: The effective component in the biological agent is an aflatoxin B1 and zearalenone degrading enzyme.

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