Detoxifying enzyme novel porin, method for preparing the same, and use thereof

Abstract

The present application relates to the technical field of biological enzymes, and particularly relates to a detoxification enzyme Novel porin as well as a preparation method and application thereof.The detoxification enzyme Novel porin is a microorganism-derived enzyme, and an amino acid sequence of the detoxification enzyme is shown as SEQ ID NO.1.The detoxification enzyme exhibits extremely high degradation activity on mycotoxins.Especially in terms of degradation efficiency on AFB1 and ZEN, the degradation efficiency is significantly better than that of microorganisms or other detoxification enzymes.The detoxification enzyme is used for degradation of mycotoxins, and has the advantages of high efficiency, short cycle, high safety, strong specificity and environmental friendliness, etc.Compared with the microbial degradation method, the mycotoxins are degraded by the detoxification enzyme, so that the harm caused by the toxicity of the microorganisms themselves can be avoided, and the change of the raw material properties and the sense organ is avoided, and the degradation efficiency is greatly improved.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological enzymes, and particularly relates to a detoxification enzyme Novel porin as well as a preparation method and application thereof. BACKGROUND

[0002] In recent decades, the contamination of food and feed by mycotoxins has been a major problem worldwide. According to the survey of the Food and Agriculture Organization (FAO) of the United Nations, nearly one-quarter of the world's food is contaminated by mycotoxins. According to a global 8-year continuous evaluation report on the contamination of food and feed by mycotoxins, 72% of the detected samples were positive for mycotoxins, and 38% of the samples were contaminated by multiple mycotoxins. Compared with single mycotoxin contamination, the contamination by multiple mycotoxins is more harmful. Aflatoxin B1 (AFB1) and zearalenone (ZEN) are two mycotoxins with the widest contamination range and the strongest toxicity, which can contaminate almost all agricultural and sideline products including corn, wheat and dried fruits. AFB1 is the most toxic member of the aflatoxin family and is one of the most dangerous natural carcinogens. It is a hepatotoxic, teratogenic and carcinogenic toxin mainly produced by filamentous fungi Aspergillus, including Aspergillus flavus and Aspergillus parasiticus. It has been reported that AFB1 is related to cancer, immunosuppression and hepatotoxicity. ZEN, a mycotoxin produced by Fusarium, is one of the most widely found mycotoxins in corn and poses a serious threat to human health. Due to the strong estrogenic activity of ZEN and its metabolites, humans may suffer from health problems due to long-term exposure to these substances. In addition, mycotoxins cause significant economic losses to food and animal husbandry every year.

[0003] Various biological, chemical and physical methods have been used to treat mycotoxin contamination. However, physical detoxification has the disadvantages of poor specificity and unstable effect, and chemical detoxification also has the problems of high cost, nutrient loss, difficulty in implementation and easy secondary pollution.

[0004] The common biological detoxification method in the prior art mainly uses microorganisms to degrade mycotoxins. Such method has a slow degradation efficiency, and due to the metabolic characteristics of microorganisms, the use of microorganisms for the degradation of mycotoxins in food is likely to introduce toxins from the source of the microorganisms themselves, or have a great impact on the sensory properties and quality of the food.

[0005] Enzymatic detoxification is a more efficient and safe method for detoxification of mycotoxins, which has a broad application prospect. However, so far, the development of enzymatic detoxification at home and abroad is extremely limited.

[0006] Therefore, the present application is proposed. SUMMARY

[0007] To solve the above technical problems, the present application provides a detoxification enzyme Novel porin, a preparation method and application thereof.

[0008] Specifically, the technical solutions of the present application are as follows:

[0009] In the first aspect, the present application provides a detoxification enzyme Novel porin.

[0010] The detoxification enzyme Novel porin is a microorganism-derived enzyme, and its amino acid sequence is shown in SEQ ID NO. 1.

[0011] The detoxification enzyme Novel porin exhibits extremely high degradation activity on mycotoxins. Especially in terms of degradation efficiency on AFB1 and ZEN, it is significantly superior to that of microorganisms or other detoxification enzymes.

[0012] In the second aspect, the present application provides a detoxification enzyme combination comprising the detoxification enzyme Novel porin and further comprising a detoxification enzyme Peroxiredoxin.

[0013] The detoxification enzyme Peroxiredoxin is also a microorganism-derived enzyme, and its amino acid sequence is shown in SEQ ID NO. 2.

[0014] The detoxification enzyme Peroxiredoxin also exhibits extremely high degradation activity on mycotoxins, and although it is not as good as the detoxification enzyme Novel porin in terms of degradation efficiency on AFB1 and ZEN, it is significantly superior to that of microorganisms or other detoxification enzymes.

[0015] The detoxification enzyme Novel porin and the detoxification enzyme Peroxiredoxin can be used in combination for the degradation of mycotoxins. Compared with the use of a single detoxification enzyme, the combination of the two can be suitable for more complex degradation systems and scene environments, and better degradation effects can be obtained.

[0016] In the third aspect, the present application provides a biological material for preparing the detoxification enzyme Novel porin or the detoxification enzyme combination, and the biological material is a gene, a recombinant vector or a recombinant bacterium.

[0017] In the present application, the gene, the recombinant vector or the recombinant bacterium comprises a gene for expressing the detoxification enzyme Novel porin, and the nucleotide sequence of the gene is shown in SEQ ID NO. 3.

[0018] Preferably, the gene, the recombinant vector or the recombinant bacterium further comprises a gene for expressing the detoxification enzyme Peroxiredoxin, and the nucleotide sequence of the gene is shown in SEQ ID NO. 4.

[0019] In a fourth aspect, the present application provides a preparation method of the detoxification enzyme Novel porin, comprising the following steps:

[0020] S1, connecting the gene sequence of the detoxification enzyme Novel porin to a pET28a(+) vector to obtain a correctly-expressed recombinant plasmid;

[0021] S2, transforming the recombinant plasmid into a bacterial strain E. coli BL21(DE3) to obtain a Novel porin protein expression bacterium induced by IPTG;

[0022] S3, expressing the detoxification enzyme Novel porin using the Novel porin protein expression bacterium.

[0023] The preparation method provided by the present application can realize stable and efficient heterologous expression of the detoxification enzyme Novel porin, thereby realizing sustainable source of the detoxification enzyme Novel porin.

[0024] Preferably, the preparation method of the detoxification enzyme Novel porin further comprises:

[0025] Step S4, purifying the expressed detoxification enzyme Novel porin using GenScript High-Affinity Ni-Charged Resin FF.

[0026] The detoxification enzyme Novel porin purified by GenScript High-Affinity Ni-Charged Resin FF can be directly used for degradation of mycotoxins and exhibits good mycotoxin degradation activity.

[0027] In a fifth aspect, the present application provides application of the detoxification enzyme, the detoxification enzyme combination, the biological material or the preparation method of the detoxification enzyme Novel porin in degradation of mycotoxins.

[0028] Preferably, the mycotoxins include aflatoxin B1 and / or zearelone.

[0029] In the present application, the temperature of the degradation is preferably 60-80℃; the pH value of the degradation is preferably 8-9; and the time of the degradation is preferably 6-30h, more preferably 6-24h.

[0030] More specifically, when degrading AFB1, the optimal degradation temperature of detoxification enzyme Novel porin and detoxification enzyme Peroxiredoxin is 80℃, the optimal degradation pH value is 9, and the optimal degradation time is 12h. When degrading ZEN, the optimal degradation temperature of detoxification enzyme Novel porin is 60℃, the optimal degradation pH value is 9, and the optimal degradation time is 20h; the optimal degradation temperature of detoxification enzyme Peroxiredoxin is 60℃, the optimal degradation pH value is 9, and the optimal degradation time is 24h.

[0031] Compared with traditional physical and chemical methods, the use of the detoxification enzyme or the detoxification enzyme combination of the present application for degrading mycotoxins has a sustainable source and is more environmentally friendly; can be active at low temperature and low pressure; can target the removal of mycotoxins; has high efficiency, short cycle, high safety, strong specificity and environmental friendliness.

[0032] Compared with traditional microbial degradation methods, the use of the detoxification enzyme or the detoxification enzyme combination of the present application for degrading mycotoxins has higher efficiency, stronger specificity and higher safety; the detoxification enzyme has a shorter acquisition cycle and lower cost; and the harm caused by the toxicity of the microorganism itself and the change in the properties and sensory of the raw materials can be avoided.

[0033] In the preferred scheme provided by the present application, the application includes: using the detoxification enzyme combination to degrade mycotoxins; the mycotoxin is aflatoxin B1; the degradation system includes Na + and Zn 2+ . The addition of Na + and Zn 2 + can significantly enhance the ability of the detoxification enzyme combination to degrade aflatoxin B1.

[0034] In another preferred scheme provided by the present application, the application includes: using the detoxification enzyme Novel porin to degrade mycotoxins; the mycotoxin is zearalenone; the degradation system includes Na + and Ca 2+ . The addition of Na + and Ca 2+ can significantly enhance the ability of the detoxification enzyme Novel porin to degrade zearalenone.

[0035] Beneficial effects:

[0036] The application provides a detoxification enzyme Novel porin and a preparation method and application thereof. The detoxification enzyme Novel porin is a microorganism-derived enzyme, and the amino acid sequence of the detoxification enzyme Novel porin is shown in SEQ ID NO. 1. The detoxification enzyme Novel porin shows extremely high degradation activity on mycotoxins. Especially in terms of degradation efficiency on AFB1 and ZEN, the degradation efficiency of the detoxification enzyme Novel porin is significantly higher than that of microorganisms or other detoxification enzymes. The detoxification enzyme Novel porin is used for degradation of mycotoxins, and has the advantages of high efficiency, short cycle, high safety, strong specificity and environmental friendliness. Compared with the microbial degradation method, the degradation of mycotoxins by the detoxification enzyme Novel porin can avoid the harm caused by the toxicity of microorganisms themselves and the change of raw material properties and sensory, and the degradation efficiency is greatly improved. BRIEF DESCRIPTION OF DRAWINGS

[0037] In order to more clearly illustrate the technical solutions in the application or prior art, the drawings needed to be used in the embodiments or prior art description will be described below.

[0038] Figure 1 Peroxiredoxin and Novel porin genes amplified by PCR from Acinetobacter nosocomialis Y1 in the embodiment 1 of the application.

[0039] Figure 2 SDS-PAGE electrophoresis maps of the Peroxiredoxin and Novel porin proteins after expression and purification, as described in the embodiment 3 of the application. Wherein A is Peroxiredoxin, and B is Novel porin.

[0040] Figure 3 The results of detoxification enzyme activity determination (degradation efficiency) in the embodiment 4, the embodiment 5 and the embodiment 6 of the application. Wherein, (a) is the results of Peroxiredoxin and Novel porin degrading AFB1 and ZEN under different time; (b) is the results of Peroxiredoxin and Novel porin degrading AFB1 and ZEN under different temperature; (c) is the results of Peroxiredoxin and Novel porin degrading AFB1 and ZEN under different pH conditions.

[0041] Figure 4 The results of detoxification enzyme stability detection in the embodiment 7 of the application, reflecting the influence of different protein inactivation methods on Peroxiredoxin and Novel porin degrading AFB1 and ZEN.

[0042] Figure 5The determination results of the influence of different ion pairs on the degradation rate in the present application embodiment 8, embodiment 9, embodiment 10, embodiment 11, comparative example 1, comparative example 2, comparative example 3 and comparative example 4.

[0043] Figure 6 The molecular weight and structural formula of AFB1 in the present application embodiment 12.

[0044] Figure 7 The molecular weight and structural formula of AFB1 degradation product in the present application embodiment 12.

[0045] Figure 8 The molecular weight and structural formula of ZEN in the present application embodiment 12.

[0046] Figure 9 The molecular weight and structural formula of ZEN degradation product in the present application embodiment 12.

[0047] Figure 10 The inhibition rate of AFB1 and ZEN and their degradation products on Vibrio fischeri in the present application embodiment 12.

[0048] Figure 11 The comparison results of the degradation efficiency of two detoxification enzymes and Acinetobacter nosocomialis Y1 on AFB1 and ZEN in the present application embodiment 13.

[0049] Figure 12 The comparison results of the removal efficiency of two detoxification enzymes and Acinetobacter nosocomialis Y1 on AFB1 in peanut meal and on ZEN in corn skin in the present application embodiment 14. DETAILED DESCRIPTION

[0050] The technical solutions provided by the present application will be described in detail below in combination with the embodiments, but they should not be understood as limiting the scope of protection of the present application. If not specifically indicated, the experimental methods used in the embodiments are conventional methods; the materials, reagents and the like used can be obtained from commercial channels.

[0051] Embodiment 1

[0052] Isolation and identification of detoxification enzyme gene and construction of expression vector.

[0053] The present embodiment obtains genes with the function of degrading mycotoxins by whole genome sequencing of Acinetobacter nosocomialis Y1 (bacterial strain preserved in China General Microbiological Culture Collection Center, preservation number: CGMCC No. 23794, preservation time: November 12, 2021) and screening. The genes Peroxiredoxin and Novel porin are amplified from the reverse transcription cDNA of the Acinetobacter nosocomialis Y1 strain, respectively. Figure 1

[0054] Among them, the primer design of Peroxiredoxin introduces a BamHI restriction site in the upstream primer and a SacI restriction site in the downstream primer. The upstream primer is 5'-CGC GGATCC ATGAGCTTGATTAATACTGAAG-3'(SEQ ID NO. 7), and the downstream primer is 5'-CCG GAGCTC TTAGATTTTACCAACTAGGTCGATAG-3'(SEQ ID NO. 8).

[0055] The primer design of Novel porin introduces a BamHI restriction site in the upstream primer and an EcoRI restriction site in the downstream primer. The upstream primer is 5'-CGC GGATCC ATGCTAGCAATTGCATCAGCTC-3'(SEQ ID NO. 5), and the downstream primer is 5'-CCG GAATTC TTAGAAGCGGTATGCTGCATG-3'(SEQ ID NO. 6).

[0056] The underlined sequences are restriction enzyme sequences. The present embodiment uses high-fidelity DNA polymerase with blunt-ended amplification products. The PCR products are purified using Magen HiPure Gel Pure DNA Kit (Magen Biotech Co., Ltd.). The purified Peroxiredoxin is digested with restriction enzymes BamHI and Sac1, and the Novel porin is digested with BamHI and EcoRI. pET 28a(+) is also linearized with BamHI and Sac1, and BamHI and EcoRI, respectively, and then purified using Magen HiPure Gel Pure DNA Kit (Magen Biotech Co., Ltd.).

[0057] ​The nucleotide sequence of Peroxiredoxin is shown as SEQ ID NO. 4, and the nucleotide sequence of Novel porin is shown as SEQ ID NO. 3. The digested Peroxiredoxin or Novel porin is ligated into the linearized pET 28a(+) vector using T4 DNA ligase (Takara), and then the ligation product is transformed into DH5a cells, and the correct expression recombinant plasmid is obtained by enzyme digestion sequencing verification.

[0058] Example 2

[0059] Heterologous expression of detoxification enzyme gene

[0060] The correct expression recombinant plasmid verified in Example 1 is transformed into strain E. coli BL21(DE3), and the single colony is screened by kanamycin. The transformed strain E. coli BL21(DE3) single colony is cultured in 10 mL LB liquid medium with a final concentration of kanamycin of 50 μg / mL overnight, inoculated into 1 L LB liquid medium (containing kanamycin) at an inoculation amount of 1%, and cultured at 37°C at 180 rpm. When the bacterial concentration OD600 value reaches 0.6, IPTG is added for induction (the final concentration of IPTG is 0.5 mM), and then induced overnight (about 16 h) at 16°C to obtain Peroxiredoxin and Novel porin protein expression bacteria.

[0061] Example 3

[0062] Purification of detoxification enzyme

[0063] The expressed Peroxiredoxin and Novel porin proteins are purified using GenScript High-Affinity Ni-Charged Resin FF. 50 mg of Peroxiredoxin and Novel porin expression bacteria are respectively centrifuged at 12000 rpm at 4°C for 20 min, the supernatant is removed, the cells are washed once with PBS, and then pH 8 lysis solution is added respectively, and lysed at room temperature for 45 min. After lysis, the cells are broken by a cell sonicator for 50 min (ultrasonic power: 300 W, working time: 2 s, stop time: 3 s). The ultrasonic broken sample is centrifuged at 12000 rpm at 4°C for 20 min, and the supernatant (crude enzyme solution) is transferred to a new centrifuge tube, and then the crude enzyme solution is transferred to a Ni 2+ column which has been washed with pH 8 washing buffer and equilibrated with lysis buffer, and then the sample is eluted with 10 mL pH 8 elution buffer.

[0064] ​The two proteins were measured by Quawell Q5000 protein analyzer, the Novel porin was 2 mg / ml, and the peroxiredoxin was 1.5 mg / ml.

[0065] The results of protein purification were detected by SDS-PAGE electrophoresis, as shown in Figure 2 Figure 2, the apparent molecular weight of Peroxiredoxin and Novel porin protein was consistent with the theoretical molecular weight, which was 20.8 kDa and 27.8 kDa, respectively.

[0066] Example 4

[0067] Detoxification enzyme activity determination

[0068] Using the detoxification enzyme purified in Example 3, the detoxification enzyme was configured into a solution with a concentration of 1 mg / mL, and then AFB1 and ZEN standards were added to the detoxification enzyme solution to make the final concentration 2 μg / mL. Incubate at 60°C and 180 rpm for 6h, 12h, and 24h, respectively. After incubation, terminate the reaction by adding an equal volume of acetonitrile, and determine the degradation rate of AFB1 and ZEN by AFB1 and ZEN detoxification enzyme in different time periods by detecting the residual amount of AFB1 and ZEN by HPLC.

[0069] The results are shown in Figure 3 Figure 3A, Novel porin can degrade 100% of AFB1 and ZEN in 24h; while the degradation rate of Peroxiredoxin to AFB1 in 24h is 100%, and the degradation rate to ZEN is 89%.

[0070] Example 5

[0071] Effect of temperature on detoxification enzyme activity

[0072] Using the detoxification enzyme purified in Example 3, the detoxification enzyme was configured into a solution with a concentration of 1 mg / mL, and then AFB1 and ZEN standards were added to the detoxification enzyme solution to make the final concentration 2 μg / mL. Incubate at 28°C, 37°C, 45°C, 55°C, 60°C, 70°C and 80°C for 24h. After incubation, terminate the reaction by adding an equal volume of acetonitrile, and determine the degradation rate of AFB1 and ZEN by AFB1 and ZEN detoxification enzyme in different time periods by detecting the residual amount of AFB1 and ZEN by HPLC.

[0073] The results are shown in Figure 3As shown in the middle B figure, with the increase of temperature, the activities of Peroxiredoxin and Novel porin gradually increased. At 60℃, the activities of Peroxiredoxin and Novel porin in degrading ZEN were the strongest. At 80℃, the activities of Peroxiredoxin and Novel porin in degrading AFB1 were the strongest. Below 45℃, Novel porin had no degradation activity on AFB1, and Peroxiredoxin had very low degradation activity on AFB1.

[0074] Example 6

[0075] Effect of pH on detoxification enzyme activity

[0076] Using the detoxification enzyme purified in Example 3, the detoxification enzyme was configured into a solution with a concentration of 1 mg / mL, and then the pH value of the detoxification enzyme solution was adjusted to 2, 3, 4, 5, 6, 7, 8, 9 and 10. AFB1 and ZEN standard were added to make the final concentration 2 μg / mL. The degradation of ZEN was carried out at 60℃, and the degradation of AFB1 was carried out at 80℃. The incubation time was 12 h. After incubation, an equal volume of acetonitrile was added to terminate the reaction, and the residual amount of AFB1 and ZEN was detected by HPLC to determine the degradation rate of AFB1 and ZEN by detoxification enzyme at different time periods.

[0077] The results are shown in Figure 3 As shown in the middle C figure, when the pH was 9, the activities of Peroxiredoxin and Novel porin in degrading AFB1 and ZEN were the strongest. The degradation rates of Peroxiredoxin and Novel porin on AFB1 were 93% and 95%, respectively, and the degradation rates on ZEN were 67% and 44%, respectively. Peroxiredoxin and Novel porin both had certain acid resistance, and had certain degradation activity at pH 4 and 5.

[0078] Example 7

[0079] Stability test of detoxification enzyme

[0080] Using the detoxification enzyme purified in Example 3, the concentration of the detoxification enzyme was adjusted to 1 mg / mL, and proteinase K and EDTA were added to the detoxification enzyme, respectively, so that the final concentrations of the above two substances were 1 mg / mL and 1 mM, respectively; at the same time, two kinds of detoxification enzymes with a concentration of 1 mg / mL were boiled in a water bath for 15 min as another treatment group; the negative control group was a PBS solution, and the positive control group was a detoxification enzyme solution with a concentration of 1 mg / mL. AFB1 and ZEN were added to the above treatment group and control group solutions, respectively, so that the final concentrations were 2 μg / mL, respectively. The degradation of ZEN was carried out at 60°C, and the degradation of AFB1 was carried out at 80°C, and the incubation time was 12 h. After incubation, the same volume of acetonitrile was added to terminate the reaction, and the residual amount of AFB1 and ZEN was detected by HPLC to determine the effect of different protein inactivation methods on the detoxification enzyme of AFB1 and ZEN.

[0081] The results are shown in Figure 4 , and the proteinase K, EDTA and boiling water treatment had no significant effect on the degradation of AFB1 and ZEN by Peroxiredoxin and Novel porin.

[0082] Example 8

[0083] This example provides a method for simultaneously degrading AFB1 using detoxification enzyme Novel porin and detoxification enzyme Peroxiredoxin. The detoxification enzyme purified in Example 3 was used, and the detoxification enzyme was configured into a solution, wherein the concentration of detoxification enzyme Novel porin was 0.1 mg / ml, and the concentration of detoxification enzyme Peroxiredoxin was 0.9 mg / ml. ZnSO4 was added to the detoxification enzyme solution so that the final concentration of Zn 2+ was 1 mM, the pH value of the solution system was adjusted to 9, and then AFB1 standard was added to the detoxification enzyme solution system so that the final concentration was 2 μg / mL. Incubation was carried out at 80°C and 180 rpm for 6 h. After incubation, the same volume of acetonitrile was added to terminate the reaction, and the residual amount of AFB1 was detected by HPLC.

[0084] The detection results are shown in Figure 5 (a) figure: the degradation rate of AFB1 was 100%.

[0085] Example 9

[0086] This example provides a method for simultaneously degrading AFB1 using detoxification enzyme Novel porin and detoxification enzyme Peroxiredoxin. All the differences between this example and Example 8 are that Na2SO4 is added to the detoxification enzyme solution so that the final concentration of Na + is 1 mM.

[0087] The detection results are shown in Figure 5The degradation rate of AFB1 was 100% as shown in Figure (a).

[0088] Comparative Example 1

[0089] This comparative example provides a method for degrading AFB1 using detoxification enzyme Novel porin and detoxification enzyme Peroxiredoxin simultaneously, which is totally different from Example 8 in that LiCI is added to the detoxification enzyme solution to make the final concentration of Li 2+ 1 mM.

[0090] The detection results are shown in Figure (a). Figure 5 The degradation rate of AFB1 was 70% as shown in Figure (a).

[0091] Comparative Example 2

[0092] This comparative example provides a method for degrading AFB1 using detoxification enzyme Novel porin and detoxification enzyme Peroxiredoxin simultaneously, which is totally different from Example 8 in that LiCI is added to the detoxification enzyme solution to make the final concentration of Li + 1 mM.

[0093] The detection results are shown in Figure (a). Figure 5 The degradation rate of AFB1 was 52% as shown in Figure (a).

[0094] Example 10

[0095] This example provides a method for degrading ZEN using detoxification enzyme Novel porin, which uses the detoxification enzyme purified in Example 3. The detoxification enzyme is configured into a solution with a concentration of 1 mg / ml. CaCI2 is added to the detoxification enzyme solution to make the final concentration of Ca 2+ 1 mM, and the pH value of the solution system is adjusted to 9. Then AFB1 standard is added to the detoxification enzyme solution system to make the final concentration 2 μg / mL. Incubation is carried out at 60°C and 180 rpm for 6 h. After the incubation, the reaction is terminated by adding an equal volume of acetonitrile, and the residual amount of AFB1 is detected by HPLC.

[0096] The detection results are shown in Figure (b). Figure 5 The degradation rate of AFB1 was 100% as shown in Figure (b).

[0097] Example 11

[0098] This example provides a method for degrading ZEN using detoxification enzyme Novel porin, which is totally different from Example 10 in that Na2SO4 is added to the detoxification enzyme solution to make the final concentration of Na + 1 mM.

[0099] The detection results are shown in Figure (b). Figure 5The degradation rate of AFB1 was 100% as shown in Figure (b).

[0100] Comparative Example 3

[0101] This comparative example provides a method for degrading ZEN using detoxification enzyme Novel porin, which is entirely different from Example 10 in that CuSO4 is added to the detoxification enzyme solution, so that the final concentration of Cu 2+ is 1 mM.

[0102] The detection results are as shown in Figure (b). Figure 5 The degradation rate of AFB1 was 71% as shown in Figure (b).

[0103] Comparative Example 4

[0104] This comparative example provides a method for degrading ZEN using detoxification enzyme Novel porin, which is entirely different from Example 10 in that LiCl is added to the detoxification enzyme solution, so that the final concentration of Li + is 1 mM.

[0105] The detection results are as shown in Figure (b). Figure 5 The degradation rate of AFB1 was 24% as shown in Figure (b).

[0106] Example 12

[0107] Safety detection of detoxification enzyme

[0108] (1) Identification of AFB1 and ZEN degradation products

[0109] The present application uses Agilent 1290 infinity II ultra-high performance liquid chromatography (UHPLC) and Q Exactive Focus Orbitrap LC-MS / MS system to determine the degradation products of AFB1 and ZEN.

[0110] Peroxiredoxin and Novel porin were used to degrade AFB1 and ZEN at a final concentration of 5 μg / mL, respectively. The degradation reaction of AFB1 and ZEN by Peroxiredoxin was carried out at 60°C for 24 h, and the degradation reaction of AFB1 and ZEN by Novel porin was carried out at 80°C for 24 h. After 24 h of degradation, an equal volume of acetonitrile was added to terminate the reaction, and the sample was centrifuged at 10,000 rpm for 10 min and then filtered through a 0.22 μm filter to obtain the degradation product for testing. The positive control was a solution of AFB1 and ZEN in PBS at a final concentration of 5 μg / mL, and the negative control was a solution of Peroxiredoxin and Novel porin in PBS. The mobile phase of UHPLC was acetonitrile and water, and the sample was detected in cation and anion modes, respectively. The maximum ion injection time of Q Exactive Focus Orbitrap LC-MS / MS system was set to 100 ms, and the ion at the automatic gain control target was established to 1 x 10 6 . The spray voltage was set to 3.5 kV, the transfer temperature was 320°C, the S-Lens level was 50, the heater temperature was 325°C, the auxiliary gas was set to 5°C, and the sheath gas was set to 36°C. The mass spectrum was obtained in full scan analysis at m / z 200-1000. All the spectral data were analyzed using Xcalibur software. Through Xcalibur software analysis, the degradation products of AFB1 degraded by Peroxiredoxin and Novel porin were the same, and the degradation products of ZEN were also the same.

[0111] The molecular weight of AFB1 in the positive ion mode was 313.07 ([M+H]+), and AFB1 was detected in the positive control and two sample groups ( Figure 6 ). Further analysis found a new peak in the two sample groups, but this peak was not detected in the positive control group and the negative control group, so it was speculated that this peak was the degradation product peak of AFB1, and its molecular weight was 287.09 ([M+H]+). After literature retrieval and comparative analysis, it was found that the degradation product of AFB1 was AFD1 ( Figure 7 ), and Peroxiredoxin and Novel porin generated AFD1 by destroying the coumarin of AFB1. The toxic groups of AFB1 include furan ring and coumarin, and the destruction of coumarin significantly reduces the toxicity of AFD1.

[0112] The molecular weight of ZEN in the negative ion mode was 317.14 ([M-H]-), and ZEN was detected in the positive control and two sample groups ( Figure 8), and further analysis found a new peak in two sample groups, but not in the positive control group and the negative control group, so it was speculated that this peak was the degradation product peak of ZEN Figure 9 ), with a molecular weight of 321.06 ([M-H]-). Literature search and comparative analysis found that the degradation product of ZEN was α-zearalanol or β-zearalanol. Further toxicity analysis showed that the degradation product of ZEN was β-zearalanol, which had significantly lower toxicity than ZEN.

[0113] (2) AFB1 and ZEN degradation product cytotoxicity analysis

[0114] The present application determines the cytotoxicity of AFB1 and ZEN degradation products by utilizing the luminescence performance of Vibrio fischeri.

[0115] 1 mg / mL of Peroxiredoxin and Novel porin were used to degrade AFB1 and ZEN to a final concentration of 5 μg / mL. The degradation reaction of AFB1 and ZEN by Peroxiredoxin was carried out at 60°C, and the degradation time was 24 h. The degradation reaction of AFB1 and ZEN by Novel porin was carried out at 80°C, and the degradation time was 24 h. The positive control was AFB1 and ZEN solution dissolved in PBS with a final concentration of 5 μg / mL, and the negative control was Peroxiredoxin and Novel porin enzyme solution dissolved in PBS. Vibrio fischeri was cultured in 2216e medium at 28°C for 24 h, then an equal volume of sample or positive control or negative control was mixed with the cultured Vibrio fischeri and cultured at 28°C. The luminescence of Vibrio fischeri was detected at 3 h, 6 h, 12 h and 24 h, respectively, to determine the health status of Vibrio fischeri and further infer the cytotoxicity of AFB1 and ZEN and their degradation products.

[0116] The results are shown in Table 1. Figure 10 By comparing the inhibition rates of AFB1 and ZEN and their degradation products on Vibrio fischeri, it was found that the inhibition rates of AFB1 and ZEN on Vibrio fischeri were significantly higher than those of their degradation products, so it was inferred that the cytotoxicity of AFB1 and ZEN degradation products was significantly lower than that of AFB1 and ZEN.

[0117] Example 13

[0118] In this example, the ability of Acinetobacter nosocomialis Y1 to degrade AFB1 and ZEN was determined, and the results were compared with the ability of two detoxification enzymes to degrade AFB1 and ZEN. The specific process is as follows:

[0119] Acinetobacter nosocomialis Y1 was inoculated into 100 mL LB medium at an inoculation amount of 0.1% and cultured for 24 h, and the expression strains of two detoxification enzymes, E. coli BL21-novel porin and E. coli BL21-peroxiredoxin, were inoculated into 100 mL LB medium at an inoculation amount of 0.1%, and expression was performed according to Example 2. The final volume of the detoxification enzymes was determined to be 100 mL. 1 mL of bacterial liquid and enzyme liquid was taken, respectively, and AFB1 and ZEN were added to make the final concentration 2 μg / mL. Y1 degraded AFB1 and ZEN at 60°C and 180 rpm for 24 h; novel porin and peroxiredoxin degraded ZEN at 60°C and 180 rpm for 24 h; novel porin and peroxiredoxin degraded AFB1 at 80°C and 180 rpm for 24 h. After degradation, an equal volume of acetonitrile was added to terminate the reaction, and the residual amount of AFB1 and ZEN was detected by HPLC.

[0120] The results are shown in Table 2. Figure 11 The degradation efficiency of the two detoxification enzymes on AFB1 and ZEN was significantly higher than that of Y1.

[0121] Example 14

[0122] 10 g of AFB1-contaminated peanut meal and 10 g of ZEN-contaminated corn skin were weighed, respectively, and 10 mL of distilled water was added to each. Subsequently, 10 mL of Y1 bacterial liquid or novel porin or peroxiredoxin enzyme liquid was added to each sample. The Y1 sample group was degraded at 180 rpm and 60°C for 24 h, the novel porin and peroxiredoxin degradation ZEN sample group was degraded at 180 rpm and 60°C for 24 h, and the novel porin and peroxiredoxin degradation AFB1 sample group was degraded at 180 rpm and 80°C for 24 h. Subsequently, 20 mL of acetonitrile was added to terminate the reaction, and the samples were centrifuged at 4.0°C and 10,000 rpm for 10 min. AFB1 and ZEN in the peanut meal and corn skin were extracted using an immunoaffinity column, and the residual amount of AFB1 and ZEN was detected by HPLC.

[0123] The results are shown in Table 3. Figure 12 The removal efficiency of the two detoxification enzymes on AFB1 in peanut meal and ZEN in corn skin was significantly higher than that of Y1.

[0124] The present application provides a biological detoxification enzyme suitable for mycotoxins AFB1 and ZEN and a coding gene thereof, and has very important significance for developing efficient AFB1 and ZEN detoxification enzyme preparation, effectively controlling AFB1 and ZEN pollution in grain and feed, reducing the safety hidden danger caused by AFB1 and ZEN, and guaranteeing the development of grain and feed safety, food safety and people's health.

[0125] The above-mentioned examples only express several embodiments of the present application, facilitate specific and detailed understanding of the technical solutions of the present application, but cannot be understood as the limitation of the patent protection scope of the invention. It should be pointed out that for ordinary skilled persons in the art, without departing from the concept of the present application, a number of modifications and improvements can be made, which belong to the protection scope of the present application.

Claims

1. Use of a combination of detoxifying enzymes for the degradation of aflatoxin B1 or for the simultaneous degradation of aflatoxin B1 and zearalenone, characterized in that, The detoxification enzyme combination comprises detoxification enzyme Novel porin and detoxification enzyme Peroxiredoxin; The amino acid sequence of the detoxification enzyme Novel porin is shown as SEQ ID NO. 1; The amino acid sequence of the detoxification enzyme Peroxiredoxin is shown as SEQ ID NO. 2; The degradation temperature is 60-80℃; the degradation pH value is 8-9; and the degradation time is 6-30h. The degraded system includes Na + and Zn 2+ .

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