Codon-optimized superoxide dismutase AfSOD gene and application of codon-optimized superoxide dismutase AfSOD gene in degradation of aflatoxin B1 and zearalenone

Abstract

The invention belongs to the technical field of enzyme engineering, and particularly relates to a codon optimized superoxide dismutase AfSOD gene and application thereof in degradation of aflatoxin B1 and zearalenone. Under the conditions that the pH is 7, the temperature is 40 DEG C and the enzyme addition amount is 10 micrograms, the degradation rate of the AFB1 reaches 99.08% within 24 hours, and the degradation rate is kept to be 80% or above within 40-80 DEG C; under the conditions that the pH is 8, the temperature is 60 DEG C and the enzyme addition amount is 20 micrograms, the degradation rate of ZEN reaches 85.1% in 24 hours, and the degradation rate is kept to be 80% or above within 50-80 DEG C; the result shows that AfSOD is a toxin degrading enzyme which can tolerate a certain degree of high temperature; the degradation rates of AFB1 and ZEN in polluted peanut powder or corn powder are 73.7% and 88.2% respectively; the invention provides a new candidate for detoxification of AFB1 and ZEN in food or feed, and has a good application prospect.

Description

Technical Field

[0001] This invention belongs to the field of enzyme engineering technology, and specifically relates to a superoxide dismutase and its applications. Background Technology

[0002] Mycotoxins are toxic secondary metabolites primarily produced by filamentous fungi such as Aspergillus, Fusarium, and Penicillium. They frequently contaminate agricultural products such as grains, nuts, and animal feed, posing a significant threat to global food security, animal productivity, and public health. According to data from the Food and Agriculture Organization of the United Nations (FAO), approximately 25% of global food crops are contaminated with mycotoxins. Even low-level exposure to mycotoxins can lead to acute or chronic toxicity, including immunosuppression, carcinogenicity, teratogenicity, and endocrine disruption. Furthermore, in actual agricultural production, certain fungal strains can simultaneously produce multiple mycotoxins, which may act synergistically, triggering complex toxicological effects.

[0003] Among numerous mycotoxins, the co-contamination of aflatoxins (AFs) and zearalenone (ZEN) is particularly prominent, causing serious health problems. AFs are secondary metabolites synthesized by various filamentous fungi of the Aspergillus genus through the polyketide pathway. They are widely present in the processing, transportation, and storage of agricultural products, mainly contaminating grains, oil crops, nuts, legumes, and spices, especially peanuts and their byproducts. Improper conditions before and after peanut harvest can lead to severe aflatoxin contamination. To date, more than 20 aflatoxins have been identified, among which AFB1 (Aflatoxin B1) is the most toxic. Its toxicity mainly exerts its effects through the covalent binding of its metabolites to DNA. AFB1 is considered one of the most potent natural hepatocarcinogens and has been classified as a Group 1 carcinogen by the International Agency for Research on Cancer (IARC). It can induce hepatocellular carcinoma (HCC), cause oxidative stress and DNA damage, and acute exposure can even lead to severe liver damage or death. ZEN is characterized by estrogen-like activity, which can interfere with reproductive function in humans and animals, causing infertility and abnormal embryonic development, especially in livestock such as pigs and cattle, resulting in significant economic losses to the livestock industry. Furthermore, due to its high heat resistance, it is difficult to completely remove it using conventional processing methods. The co-contamination of these two toxins not only amplifies their individual toxicological effects but may also trigger complex health problems such as immunosuppression and endocrine disorders through mechanisms such as enhanced oxidative stress and synergistic DNA damage, posing a serious challenge to food safety.

[0004] Currently, various methods have been developed for detoxifying or removing products contaminated with mycotoxins, including physical methods (such as adsorption and heat treatment), chemical methods (such as oxidation and alkali treatment), and biological methods utilizing microorganisms or enzymes. In recent years, biological detoxification technology has become a research hotspot due to its environmental friendliness and high specificity. However, existing enzymatic hydrolysis technologies still have many structural defects, making it difficult to meet actual production needs. The primary problem is a narrow degradation spectrum. Most of the currently identified mycotoxin-degrading enzymes can only target a single toxin. For example, patent 202311691221.3 discloses a ZEN-degrading enzyme, which has a strong degradation ability against the single fungal toxin ZEN, but cannot be applied to environments with multiple toxins co-contaminated. A few studies have reported enzymes that can simultaneously degrade two toxins, but these generally suffer from low activity and demanding reaction conditions, making industrial application difficult. Therefore, finding and identifying new broad-spectrum enzymes, especially novel enzymes that degrade AFB1 and ZEN, is crucial. Summary of the Invention

[0005] To address the aforementioned issues, this invention proposes a codon-optimized superoxide dismutase (AfSOD) gene and its application in the degradation of aflatoxin B1 and zearalenone.

[0006] The technical solution of this invention is implemented as follows: This invention provides a codon-optimized superoxide dismutase AfSOD gene. The nucleotide sequence of the superoxide dismutase AfSOD encoding gene, as shown in SEQ ID No. 2, is optimized using E. coli codons, and the optimized sequence is shown in SEQ ID No. 3.

[0007] Preferably, the amino acid sequence of the protein encoded by the codon-optimized superoxide dismutase AfSOD gene is shown in SEQ ID No. 1.

[0008] Secondly, a recombinant plasmid containing the codon-optimized superoxide dismutase AfSOD gene described above.

[0009] Thirdly, a recombinant strain containing the aforementioned recombinant plasmid.

[0010] Fourthly, the application of the codon-optimized superoxide dismutase AfSOD gene, the recombinant plasmid, or the recombinant strain described above in the degradation of aflatoxin B1 (AFB1) and / or zearalenone (ZEN).

[0011] Preferably, the degradation products of aflatoxin B1 are AFQ1 or epi-AFQ1, and the degradation products of zearalenone are 13-OH-ZEN or 15-OH-ZEN.

[0012] Preferably, the degradation temperature is 20-80℃ and the pH is 4.0-10.0.

[0013] Under optimal conditions of pH 7.0, temperature 40℃, and enzyme addition of 10 μg, the degradation rate of AFB1 reached 99.08% after 24 h, and maintained a degradation rate of over 80% within the range of 40-80℃.

[0014] Under optimal conditions of pH 8.0, temperature 60℃, and enzyme addition of 20 μg, the degradation rate of ZEN reached 85.1% after 24 h, and maintained a degradation rate of over 80% within the range of 50-80℃.

[0015] Fifthly, the application of the codon-optimized superoxide dismutase AfSOD gene, the recombinant plasmid, or the recombinant strain mentioned above in cereal detoxification.

[0016] Preferably, the grains mentioned above are peanuts and corn, and the detoxification process involves the removal of AFB1 and / or ZEN.

[0017] Preferably, the concentration of superoxide dismutase AfSOD used during the above detoxification process is 50 μg / mL-100 μg / mL.

[0018] The present invention has the following beneficial effects: This invention provides a superoxide dismutase (AfSOD) capable of simultaneously degrading AFB1 and ZEN. Under optimal conditions of pH 7.0, temperature 40°C, and enzyme dosage of 10 μg, the degradation rate of AFB1 reaches 99.08% after 24 h, and maintains a degradation rate of over 80% within the temperature range of 40-80°C. Under optimal conditions of pH 8.0, temperature 60°C, and enzyme dosage of 20 μg, the degradation rate of ZEN reaches 85.1% after 24 h, and maintains a degradation rate of over 80% within the temperature range of 50-80°C. This indicates that AfSOD is a fungal toxin degrading enzyme that can withstand a certain degree of high temperature. The molecular formula of the AFB1 degradation product is C1. 17 H 12 O7 and C 17 H 14 O8, ZEN degradation products have the molecular formula C 18 H 20 O6 and C 18 H 22Based on the structures of AFB1 and ZEN, the degradation products of the two toxins are identified as AFQ1 / epi-AFQ1 and 13-OH-ZEN / 15-OH-ZEN, respectively. This application demonstrated that by adding different concentrations of AfSOD (50 and 100 μg / mL) to contaminated peanut flour or corn flour samples and incubating them at 50℃ and 150 rpm for 6–24 h, the degradation rates of AFB1 and ZEN were 73.7% and 88.2%, respectively. Furthermore, zebrafish experiments further confirmed that AfSOD has no significant toxicity to the degradation products of AFB1 and ZEN. This application provides a new candidate for the detoxification of AFB1 and ZEN in food or feed, and has promising application prospects. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 The plasmid map is for pET-28a(+).

[0021] Figure 2 The recombinant plasmid map of pET-28a(+)-AfSOD.

[0022] Figure 3 This is an SDS-PAGE validation image of superoxide dismutase AfSOD, where M: standard protein marker; P: purified protein.

[0023] Figure 4 The degradation rates of AFB1 and ZEN by superoxide dismutase AfSOD under different metal ion conditions are shown.

[0024] Figure 5 The degradation rates of AFB1 and ZEN by superoxide dismutase AfSOD under different pH conditions are shown.

[0025] Figure 6 The degradation rates of AFB1 and ZEN by superoxide dismutase AfSOD under different temperature conditions are shown.

[0026] Figure 7 The degradation rates of AFB1 and ZEN were measured by different amounts of superoxide dismutase (AfSOD) added.

[0027] Figure 8 The degradation rates of AFB1 and ZEN by superoxide dismutase AfSOD at different reaction times are shown.

[0028] Figure 9 The results are HPLC analysis of the degradation of AFB1 in peanut powder contaminated with AFB1 by superoxide dismutase (AfSOD); where A: control group without superoxide dismutase (AfSOD), B: treatment group with superoxide dismutase (AfSOD) added at 50 μg / mL, and C: treatment group with superoxide dismutase (AfSOD) added at 100 μg / mL.

[0029] Figure 10 The results of HPLC analysis on the degradation of ZEN-contaminated corn flour by superoxide dismutase (AfSOD) are shown below: A: Control group without superoxide dismutase (AfSOD); B: Treatment group with superoxide dismutase (AfSOD) at a concentration of 50 μg / mL; C: Treatment group with superoxide dismutase (AfSOD) at a concentration of 100 μg / mL.

[0030] Figure 11 This is a secondary mass spectrum of the degradation products of AFB1 by superoxide dismutase AfSOD; where A represents degradation product C. 17 H 12 The mass spectrum of O7, B is the degradation product C. 17 H 14 Mass spectrum of O8.

[0031] Figure 12 This is a secondary mass spectrum of the degradation products of ZEN by superoxide dismutase AfSOD; where A is the degradation product C. 18 H 20 The mass spectrum of O6, B is the degradation product C. 18 H 22 Mass spectrum of O6.

[0032] Figure 13 Toxicity analysis of AFB1 and ZEN degradation products after AfSOD treatment; where A represents the effect of AFB1 and its degradation products on zebrafish morphology and liver fluorescence intensity, B represents the effect of ZEN and its degradation products on zebrafish morphology, and C represents the effect of degradation products on zebrafish liver fluorescence intensity. Detailed Implementation

[0033] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0034] Unless otherwise specified, the experimental methods used in the following experimental examples are conventional methods; the materials and reagents used are commercially available unless otherwise specified.

[0035] Superoxide dismutase (SOD) is a metal-binding antioxidant enzyme that catalyzes the conversion of superoxide anion radicals into oxygen and hydrogen peroxide, thereby protecting cells from oxidative stress damage. Therefore, SOD is considered a major intracellular detoxification enzyme and a core component of the antioxidant defense system. Due to its strong antioxidant, anti-inflammatory, anti-aging, and membrane-stabilizing effects, commercially available SOD has broad application prospects in pharmaceuticals, food technology, agricultural science, and cosmetics. Although the classic function of SOD is primarily focused on combating oxidative stress, increasing evidence suggests it may possess a wider range of biochemical functions.

[0036] Biomaterials: Strains: Escherichia coli DH5α, Escherichia coli BL21(DE3), purchased from Sangon Biotech (Shanghai) Co., Ltd.

[0037] Culture medium: LB medium: 10 g·L tryptone, 5 g·L yeast extract, 10 g·L sodium chloride, pH 7.0-7.2.

[0038] Experimental reagents: AFB1 and ZEN standard samples were purchased from Sigma-Aldrich; other reagents were domestically produced analytical grade.

[0039] Example 1: Preparation of recombinant Escherichia coli BL21(DE3) The superoxide dismutase (AfSOD) encoding gene, with the nucleotide sequence shown in SEQ ID No. 2, was synthesized by Sangon Biotech (Shanghai) Co., Ltd. Codon optimization was performed based on the codon preferences of *E. coli* BL21(DE3) to achieve efficient expression of the exogenous gene in *E. coli*. This invention optimized the codons of the target gene because the codon usage preferences of the original gene source species differ significantly from those of *E. coli*. Direct expression would lead to translational arrest, low expression levels, or protein misfolding. By optimizing the codons to those preferred by *E. coli*, mRNA stability and translation efficiency can be significantly improved, thereby increasing protein expression and obtaining recombinant proteins with correct conformation and activity. Therefore, codon optimization is a necessary measure to achieve the technical effects of this invention. The optimized sequence is shown in SEQ ID No. 3, with a restriction enzyme site Sac1 introduced at the 5' end and a restriction enzyme site Not I introduced at the 3' end.

[0040] The PCR amplification system and amplification program are shown in Tables 1 and 2: Table 1 PCR amplification system Table 2 PCR amplification program The amino acid sequence of the superoxide dismutase AfSOD protein is shown in SEQ ID No. 1. The gene sequence with the nucleotide sequence shown in SEQ ID No. 3 was ligated into the *E. coli* expression vector pET-28a(+) (…). Figure 1 At the corresponding site, the recombinant plasmid pET-28a(+)DPPⅢ (as shown) was obtained. Figure 2 (As shown). The obtained recombinant plasmid was added to E. coli DH5a competent cells, heat-shocked in a 42°C water bath for 45 seconds, and then immediately placed on ice to cool for 2-3 minutes for transformation of the recombinant product. For the correctly transformed strains, the plasmid was extracted and introduced into the E. coli expression strain BL21(DE3) by heat shock. Positive clones were screened by LB agar containing kanamycin and verified by bacterial PCR to obtain recombinant E. coli that successfully expressed superoxide dismutase AfSOD.

[0041] Example 2: Induction and purification of superoxide dismutase AfSAOD 1. Inducible expression of superoxide dismutase AfSAOD Escherichia coli BL21(DE3) / pET-28a(+)-AfSAOD was inoculated into 25 mL LB medium and cultured at 37°C with shaking at 200 rpm for 12 h. Then, 2% of the culture was inoculated into 300 mL LB liquid medium and cultured at 37°C with shaking at 200 rpm until OD500 reached its maximum. 600 =0.6~0.8, add IPTG to a final concentration of 0.1 mM, and induce at 16℃ for 20 h.

[0042] 2. Purification of superoxide dismutase AfSAOD Collect the induced bacterial culture, centrifuge at 8000 rpm for 10 min at 4°C, and discard the supernatant. Resuspend the bacterial cells in 10 mL of equilibration buffer, sonicate for 30 min under ice bath conditions (sonication power 300W, sonication on for 4 s, sonication off for 4 s), centrifuge at 12000 rpm for 10 min at 4°C, collect the supernatant and filter it through a 0.45 μm aqueous filter membrane. Then, purify the protein using a nickel-NTA column with a prepared 200 mM imidazole elution buffer, collect the eluent, and ultrafilter the eluent through a 10 kDa ultrafiltration tube at 4000 rpm for 30 min at 4°C to remove imidazole, obtaining the purified protein. The purification effect of the purified protein was detected by SDS-PAGE gel electrophoresis, and the results are shown below. Figure 3 As shown, the purified AfSOD exhibited a single, clear band on SDS-PAGE, with a molecular weight between 25-30 kDa, consistent with the predicted molecular weight.

[0043] Example 1: Degradation of AFB1 by the purified superoxide dismutase AfSAOD prepared in Example 2. The protein purified in Example 2 was analyzed for protein concentration using a Bradford protein assay kit and stored at 4°C for later use. The degradation of AFB1 by superoxide dismutase (AfSAOD) was carried out in a 500 μL system containing 5.0 μg / mL AFB1, 20 μg AfSAOD protein, and 50 mM Tris-HCl buffer (containing 0.5 mM metal ions, pH 7.0). After co-incubation for 24 h, the reaction was terminated by adding 500 μL of methanol. The system was thoroughly mixed and filtered through a 0.22 μm organic filter membrane. The AFB1 content was determined by high-performance liquid chromatography (HPLC).

[0044] The chromatographic conditions for detecting AFB1 using high performance liquid chromatography are as follows: Chromatographic column: Proshe11 120-C18 column (150×4.6 mm, 4 μm); UV detector: detection wavelength 365 nm; flow rate: 1 mL / min; injection volume: 10; acquisition time: 10 min; column temperature: 35℃; mobile phase: methanol:acetonitrile:water = 1:1:3 (v / v / v).

[0045] The AFB1 degradation rate is calculated using the following formula: Degradation rate = (AFB1 content in control group - AFB1 content in experimental group) / AFB1 content in control group The pH of the reaction system was adjusted to 7.0, the temperature was controlled at 40℃, and 0.5 mM Zn was added. 2＋ Fe 3＋ Ni ＋ Mn 2＋ Mg 2＋ Cu 2＋ K ＋ ,Depend on Figure 4 It can be seen that in Cu 2＋ When present, superoxide dismutase AfSOD degrades AFB1.

[0046] In enzymatic reactions, the pH and temperature of the reaction system significantly affect enzyme activity. In this example, the effects of varying the pH and temperature of the reaction system on the degradation rate of AFB1 by superoxide dismutase (AfSOD) were investigated. After determining the optimal pH and temperature, the amount of AfSOD added was optimized accordingly. The specific steps are as follows: Adjust the pH of the reaction system to 4.0-10.0 and control the temperature at 40℃. Figure 5 It can be seen that the degradation rate of AFB1 is the highest at pH 7.0, with a degradation rate of 96.95% after 24 h.

[0047] The reaction temperature was adjusted to 20-80℃ at pH=7.0. Figure 6 It can be seen that the degradation rate of AFB1 reaches its highest at 40℃, and maintains a degradation rate of over 80% in the range of 40-80℃.

[0048] The pH of the reaction system was adjusted to 7.0, and the temperature was controlled at 40℃. The amounts of superoxide dismutase (AfSOD) added were 5, 10, 15, 20, 30, 40, and 50 μg, respectively. The results are as follows: Figure 7 As shown, the degradation rate of AFB1 by superoxide dismutase AfSOD increases with the increase of the amount of superoxide dismutase AfSOD, but the increasing trend gradually slows down. From an economic point of view, the optimal enzyme amount is 10 μg.

[0049] The degradation rate of AFB1 at different reaction times was investigated under the conditions of pH 7.0, temperature 40℃, and enzyme addition of 10 μg. The results are as follows: Figure 8 As shown, the degradation rate of AFB1 increases with time, reaching 99.08% at 24 h.

[0050] Example 2: Degradation of ZEN by purified superoxide dismutase AfSOD prepared in Example 2 The degradation reaction of ZEN by superoxide dismutase (AfSOD) was carried out in a 500 L system containing 10 μg / mL ZEN, 20 μg AfSOD protein, and 50 mM Tris-HCl buffer (containing 0.5 mM metal ions, pH 7.0). After incubation for 24 h, 500 μL of methanol was added to terminate the reaction. The system was thoroughly mixed and filtered through a 0.22 μm organic filter membrane. The ZEN content was determined by high performance liquid chromatography (HPLC).

[0051] The chromatographic conditions for detecting ZEN using high performance liquid chromatography are as follows: Column: Proshe11 120-C18 column (150×4.6 mm, 4 μm); Fluorescence detector: excitation wavelength 235 nm, emission wavelength 470 nm; Flow rate: 1 mL / min; Injection volume: 10 L; Acquisition time: 5 min; Column temperature: 35℃; Mobile phase: methanol:water = 4:1 (v / v).

[0052] The ZEN degradation rate is calculated using the following formula: Degradation rate = (ZEN content in control group - ZEN content in experimental group) / ZEN content in control group The pH of the reaction system was adjusted to 7.0, the temperature was controlled at 40℃, and 0.5 mM Zn was added. 2＋ Fe 3＋ Ni ＋ Mn 2＋ Mg 2＋ Cu 2＋ K ＋ .Depend on Figure 4 It can be seen that in Cu 2＋ When present, superoxide dismutase (AfSOD) exhibits the best degradation effect on ZEN.

[0053] Adjust the pH of the reaction system to 5.0-10.0 and control the temperature at 40℃. Figure 5 It can be seen that the degradation rate of ZEN is the highest at pH 8.0, and the degradation rate is 95% after 24 h.

[0054] The reaction temperature was adjusted to 20-80℃ at pH=8.0. Figure 6 It can be seen that the degradation rate of ZEN reaches its highest at 60℃, and maintains a degradation rate of over 80% in the range of 50-80℃.

[0055] The pH of the reaction system was adjusted to 8.0, and the temperature was controlled at 60℃. The amounts of superoxide dismutase (AfSOD) added were 5, 10, 15, 20, 30, 40, and 50 μg, respectively. The results are as follows: Figure 7 As shown, the degradation rate of ZEN by superoxide dismutase AfSOD increases with the increase of the amount of superoxide dismutase AfSOD, but the increasing trend gradually slows down. From an economic point of view, the optimal enzyme amount is 50 μg.

[0056] The degradation rate of ZEN at different reaction times was investigated under the conditions of pH 8.0, temperature 60℃, and enzyme addition of 50 μg. The results are as follows: Figure 8 As shown, the degradation rate of ZEN increases with time, reaching 85.1% at 24 h.

[0057] Example 3: Analysis of the degradation products of AFB1 and ZEN by the purified superoxide dismutase AfSOD prepared in Example 2. To analyze the degradation products, AfSOD was incubated with 1 μg / mL AFB1 and 1 μg / mL ZEN under optimal degradation conditions for 24 h. The degradation products were analyzed using UPLC-MS / MS. Chromatographic separation was performed using a reversed-phase CORTECS™ UPLC C18 column (100 × 2.1 mm, 1.6 μm), with a mobile phase of methanol and water containing 0.1% formic acid, at a flow rate of 0.3 mL / min. Mass spectrometry detection was performed using positive ion electrospray ionization (ESI) mode, with a scan mass range of 100–800 m / z. The spray voltage was set to 3500 V, the nitrogen flow rate to 10 L / min, and the probe heating temperature to 350 °C. The analysis and identification of the degradation products were performed using CompoundDiscoverer 3.3 software. Figure 11 It can be seen that the molecular formula of the AFB1 degradation product is C 17 H 12 O7 and C 17 H 14 O8, by Figure 12 It can be seen that the molecular formula of ZEN degradation products is C 18 H 20 O6 and C 18 H 22 O6. Based on the structures of AFB1 and ZEN, it is speculated that the degradation products of the two toxins are AFQ1 / epi-AFQ1 and 13-OH-ZEN / 15-OH-ZEN, respectively.

[0058] Example 4: Toxicity analysis of purified superoxide dismutase AfSOD prepared in Example 2 on AFB1 and ZEN degradation products. Based on previous studies, zebrafish toxicology models have proven reliable in assessing early developmental toxicity and detecting the potential toxicity of mycotoxin degradation products. In this study, wild-type AB zebrafish and transgenic zebrafish (tg) were purchased from Nanjing Ezerica Biotechnology Co., Ltd. (Nanjing, China). Based on preliminary experimental results, the working concentrations of AFB1 and ZEN were determined to be 80 ng / mL and 2 μg / mL, respectively. Six groups of zebrafish larvae (10 fish per group) were placed in different well plates. The first three groups were treated with AFB1: (a) control group, (b) 80 ng / mL AFB1 degradation product group, and (c) 80 ng / mL AFB1 group. The remaining three groups were treated with ZEN: (I) control group, (II) 2 μg / mL ZEN degradation product group, and (III) 2 μg / mL ZEN group. The incubation temperature was set at 28°C, and the culture medium was changed every 24 h to maintain culture conditions and compound stability. The zebrafish liver fluorescence intensity was quantitatively analyzed using a Leica microscope at 50× magnification and ImageJ software.

[0059] The results are as follows Figure 13 As shown, zebrafish were used in this study to further verify the detoxification effects of AfSOD-mediated AFB1 and ZEN degradation products. Exposure to 80 ng / mL AFB1 or 2 μg / mL ZEN in the treatment groups induced significant developmental abnormalities, including spinal curvature and yolk sac edema, accompanied by decreased liver fluorescence intensity, indicating liver damage. In contrast, zebrafish larvae in the 80 ng / mL AFB1 degradation product group and the 2 μg / mL ZEN degradation product group did not show obvious morphological abnormalities or liver damage, and their toxicity was significantly lower than that of the maternal toxin. This indicates that the degradation products of AfSOD have low toxicity and demonstrate their potential for safe application in food and feed systems.

[0060] Application example: Degradation of AFB1 and ZEN in peanut flour and corn flour contaminated with superoxide dismutase (AfSOD). Peanut and corn samples were ground and passed through an 80-mesh sieve. Then, 5 g of peanut powder or corn powder was added to 50 mL of distilled water and placed in a 100 mL Erlenmeyer flask, which was sterilized at 121 °C for 20 minutes. After cooling, AFB1 (final concentration 5 μg / mL) and ZEN (final concentration 10 μg / mL) were added to the peanut and corn samples respectively for artificial contamination. Different concentrations of AfSOD (50 and 100 μg / mL) were then added to the contaminated samples. The mixtures were incubated at 50 °C and 150 rpm for 24 h with shaking. To determine the optimal incubation time, the enzyme concentration was incubated at 100 μg / mL for 6, 12, and 24 h, respectively. After the reaction, the mixture was sonicated and centrifuged to obtain the supernatant. The toxin was then extracted using 50 mL of dichloromethane. After drying under nitrogen, the residue was redissolved in 1 mL of methanol, and the toxin residue was analyzed by HPLC. Figure 9 , Figure 10 It can be seen that the degradation rates of AFB1 and ZEN in peanut flour contaminated with AFB1 and corn flour contaminated with ZEN were 73.7% and 88.2%, respectively.

[0061] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A codon-optimized superoxide dismutase AfSOD gene, characterized in that: The superoxide dismutase AfSOD gene with the nucleotide sequence shown in SEQ ID No. 2 was optimized using E. coli codons, and the optimized sequence is shown in SEQ ID No.

3.

2. The codon-optimized superoxide dismutase AfSOD gene according to claim 1, characterized in that: The amino acid sequence of the protein encoded by the codon-optimized superoxide dismutase AfSOD gene is shown in SEQ ID No.

1.

3. A recombinant plasmid containing the codon-optimized superoxide dismutase AfSOD gene as described in claim 1.

4. A recombinant bacterial strain, characterized in that: Contains the recombinant plasmid as described in claim 3.

5. The use of the codon-optimized superoxide dismutase AfSOD gene of claim 1, the recombinant plasmid of claim 3, or the recombinant strain of claim 4 in the degradation of aflatoxin B1 and / or zearalenone.

6. The application according to claim 5, characterized in that: The degradation products of aflatoxin B1 are AFQ1 or epi-AFQ1, and the degradation products of zearalenone are 13-OH-ZEN or 15-OH-ZEN.

7. The application according to claim 6, characterized in that: The degradation temperature is 20-80℃, and the pH is 4.0-10.

0.

8. The application of the codon-optimized superoxide dismutase AfSOD gene of claim 1, the recombinant plasmid of claim 3, or the recombinant strain of claim 4 in cereal detoxification.

9. The application according to claim 8, characterized in that: The grains are peanuts and corn, and the detoxification process involves the removal of aflatoxin B1 and / or zearalenone.

10. The application according to claim 9, characterized in that: The concentration of superoxide dismutase (AfSOD) used during detoxification is 50-100 μg / mL.

Images