A peroxidase mutant m3 with improved thermal stability and its application in degrading mycotoxins

Abstract

The application belongs to the technical field of bioengineering and feed safety, and particularly relates to a peroxidase mutant M3 with improved thermal stability and application thereof in degrading mycotoxins. The mutant M3 obtained in the application can efficiently degrade AFB1 and ZEN, and the degradation rates thereof are 98.91% and 97.69% respectively under the condition of 50 DEG C. The half-life of M3 is increased to 14.73 h from 6.87 h of the wild type under the condition of 50 DEG C, which is about 2.14 times of the wild type, indicating that the thermal stability of M3 is higher. The degradation products have good biological safety, and still have high detoxification efficiency in actual food or feed matrix. The mutant realizes the synergistic optimization of thermal stability and catalytic performance, and provides a basis for the application of BVDyp in industrial mycotoxin detoxification.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering and feed safety technology, and specifically relates to a peroxidase mutant and its application in degrading fungal toxins. Background Technology

[0002] Mycotoxins are a class of toxic secondary metabolites produced by fungi during their growth and reproduction. They are widely found in grains, oil crops, nuts, feed, and their processed products. Due to their characteristics of high concealment of contamination, wide range of contamination, stable physicochemical properties, and significant toxicity, mycotoxins have become one of the most important issues facing global food and feed safety. Among the many mycotoxins, aflatoxin B1 (AFB1) and zearalenone (ZEN) have attracted much attention due to their high incidence, high toxicity, and serious harm to human and animal health. AFB1, mainly produced by toxin-producing fungi such as Aspergillus flavus and Aspergillus parasiticus, is one of the most toxic natural mycotoxins known, exhibiting significant hepatotoxicity, mutagenicity, carcinogenicity, and immunosuppressive effects. ZEN, mainly produced by Fusarium fungi, widely contaminates corn, wheat, and their feed products. It has significant estrogen-like activity and can cause reproductive dysfunction, endocrine imbalance, and decreased production performance in animals. AFB1 and ZEN often contaminate agricultural products and feed ingredients, either alone or in combination, and may persist during storage, transportation, and processing, thus posing a long-term threat to agricultural production, livestock farming, the food industry, and human health.

[0003] Currently, methods for controlling or detoxifying AFB1 and ZEN mainly include physical, chemical, and biological methods. Physical methods typically include screening and grading, adsorption removal, irradiation treatment, heat treatment, and ozone treatment. These methods are relatively simple to operate, but often suffer from limited removal efficiency, high processing costs, adverse effects on nutritional components and sensory quality, and difficulty in completely removing toxins. In particular, AFB1 and ZEN have strong chemical stability and are not easily completely inactivated under conventional processing and heat treatment conditions; therefore, relying solely on physical treatment methods is usually insufficient to meet the needs of deep detoxification. Chemical methods mainly destroy the toxin structure through oxidation, reduction, acid-base treatment, or the addition of chemical detoxifying agents, thereby reducing their toxicity. This type of method can achieve high detoxification efficiency under certain conditions, but it also has significant drawbacks. For example, chemical reagent residues may pose new safety risks; the processing conditions are relatively harsh and can easily affect the nutritional quality of food or feed; at the same time, whether the products of chemical treatment are completely non-toxic often still needs further verification. Therefore, the application of chemical methods in food and feed is somewhat limited.

[0004] In contrast, biological detoxification technology has received increasing attention in recent years due to its advantages such as mild conditions, high specificity, minimal impact on nutritional quality, and environmental friendliness. Biological methods mainly include using microbial cells to adsorb toxins, using microbial metabolism to transform toxins, and using enzyme preparations to catalyze the degradation of toxins. Among these, enzymatic detoxification is considered one of the most promising technical routes due to its high reaction specificity, high catalytic efficiency, ease of process control, and suitability for standardized preparation and application. Especially for mycotoxins like AFB1 and ZEN, which have different structures and toxicological characteristics, obtaining an enzyme preparation with broad-spectrum degradation capabilities and high safety could significantly improve the efficiency and practical application value of mycotoxin control.

[0005] Peroxidases are a class of oxidoreductases that catalyze the oxidation of various substrates using hydrogen peroxide as an oxidant. They have broad application potential in lignin conversion, dye decolorization, environmental pollutant removal, and food safety. Dye-decolorizing peroxidases (DyPs) typically use heme as a cofactor, exhibiting a broad substrate spectrum and strong oxidizing capacity. BVDyp belongs to the DyP-type peroxidase and has the potential to degrade AFB1 and ZEN. However, existing peroxidases still have certain shortcomings when used for mycotoxin detoxification: First, some enzymes can only effectively act on a single toxin, making it difficult to meet the detoxification requirements under conditions of complex pollution; second, some enzymes are easily inactivated during high-temperature or long-term treatment, which is detrimental to formulation storage, transportation, and industrial application; third, the catalysis of peroxidases depends on H2O2, and an unsuitable H2O2 concentration may cause oxidative inactivation of enzyme molecules; fourth, complex food or feed matrices may reduce the actual detoxification efficiency of the enzymes. For example, the invention patent with publication number CN111073867A discloses a dye decolorizing peroxidase BsDyP, which can efficiently degrade various mycotoxins such as zearalenone and aflatoxin. However, its optimal catalytic temperature is only 42℃, which is not suitable for industrial high-temperature detoxification scenarios. In addition, current industry modifications to dye decolorizing peroxidases mostly focus on optimizing degradation efficiency, lacking targeted thermal stability modifications for high-temperature mycotoxin detoxification scenarios.

[0006] Thermostability of enzymes is one of the key indicators affecting their industrial application performance. During feed processing, raw material storage, formulation preparation, and transportation, enzyme preparations typically need to withstand certain temperature conditions. Insufficient thermostability leads to a rapid decline in enzyme activity, thereby reducing detoxification efficiency and increasing application costs. Therefore, further improving the thermostability of mycotoxin-degrading enzymes while maintaining or enhancing catalytic activity to achieve efficient and low-cost industrial detoxification applications of mycotoxins is a crucial problem that urgently needs to be solved. Summary of the Invention

[0007] This invention proposes a peroxidase mutant M3 with improved thermal stability and its application in the degradation of mycotoxins, which solves the problems of insufficient thermal stability and limited degradation efficiency in practical matrices of existing mycotoxin degrading enzymes.

[0008] The technical solution of this invention is implemented as follows:

[0009] This invention provides a peroxidase BVDyp mutant M3 with improved thermal stability and AFB1 and ZEN degradation activity. This invention also provides the coding gene, expression vector, engineered bacteria, preparation method, and its application in the detoxification of peanut flour, corn flour, feed, or other substrates contaminated with fungal toxins.

[0010] On the one hand, the present invention provides a peroxidase mutant M3 with improved thermal stability, the amino acid sequence of which is shown in SEQ ID NO.1.

[0011] Preferably, the mutant M3 is obtained by mutating threonine at position 46 of the wild-type peroxidase BVDyp, as shown in SEQ ID NO.3, to proline, alanine at position 82 to leucine, and cysteine ​​at position 405 to isoleucine.

[0012] After treatment at 50℃, the relative enzyme activities of wild-type, M1, M2, and M3 were 36.0±2.0%, 51.10±1.2%, 52.7±1.2%, and 77.10±2.0%, respectively. After treatment at 60℃, the relative enzyme activities of M1, M2, and M3 increased by 18.2±1.0%, 20.5±2.0%, and 48.5±1.2% compared to the wild-type, respectively. The half-life of mutant M3 at 50℃ reached 14.73 h, while the half-life of wild-type BVDyp was 6.87 h, approximately 2.14 times that of the wild-type, indicating that it has superior thermostability.

[0013] Secondly, the present invention also provides the encoding gene of the above-mentioned peroxidase mutant M3, the nucleotide sequence of which is shown in SEQ ID NO.2.

[0014] Thirdly, a recombinant vector expressing the above-mentioned peroxidase mutant M3 contains the above-mentioned encoding gene.

[0015] Fourthly, a recombinant strain expressing the above-mentioned peroxidase mutant M3 contains the above-mentioned coding gene or contains the above-mentioned recombinant vector.

[0016] A method for constructing a peroxidase BVDyp mutant includes the following steps:

[0017] 1) Obtain the amino acid sequence and / or three-dimensional structure information of wild-type BVDyp;

[0018] 2) Predicting protein structure based on AlphaFold3;

[0019] 3) FoldX was used to score the stability of candidate mutation sites;

[0020] 4) Conduct a conservatism and substitutability analysis using PSSM;

[0021] 5) Screening yields the combination of target mutation sites;

[0022] 6) Obtain the BVDyp mutant of peroxidase through site-directed mutagenesis;

[0023] 7) Express, purify and verify the performance of the obtained mutants.

[0024] Preferably, the target mutation site combination is T46P / A82L / C405I.

[0025] Fifthly, this invention claims protection for the application of the above-mentioned peroxidase mutant M3, wherein the application includes any one of the following:

[0026] (1) Application in the degradation of mycotoxins;

[0027] (2) Application in the preparation of fungal toxin detoxification agents;

[0028] (3) Application in detoxification of food, feed or agricultural products;

[0029] (4) Application in improving the thermal stability of peroxidase.

[0030] M3 can degrade AFB1 and ZEN in the range of 20-80℃, with degradation rates of 98.91% and 97.69% for the two toxins, respectively, at 50℃.

[0031] Preferably, the aforementioned mycotoxin is AFB1 and / or ZEN, wherein the degradation product of AFB1 is AFQ1, and the degradation product of ZEN is 15-OH-ZEN.

[0032] Sixthly, this invention also claims protection for a method to improve the thermal stability of peroxidase, which involves site-directed mutagenesis using wild-type peroxidase BVDyp with the amino acid sequence shown in SEQ ID NO.3 as the parent, including at least one of mutating threonine at position 46 to proline, alanine at position 82 to leucine, and cysteine ​​at position 405 to isoleucine, to obtain a peroxidase with improved thermal stability.

[0033] Seventhly, this invention claims a method for removing fungal toxins from a food matrix, wherein the above-mentioned peroxidase mutant M3 is mixed with the food matrix to be treated, and then H2O2 is added to carry out a detoxification reaction.

[0034] Preferably, the food matrix is ​​peanut flour or corn flour, and the mycotoxin is AFB1 and / or ZEN. M3 achieves a degradation rate of 66.81% for AFB1 in peanut flour and a degradation rate of 63.39% for ZEN in corn flour.

[0035] Preferably, the concentration of the peroxidase mutant M3 is 20-30 μg / mL, and the final concentration of H2O2 is 0.1 mmol / L.

[0036] The present invention has the following beneficial effects:

[0037] 1. The BVDyp three-point mutant M3 provided by this invention can efficiently degrade AFB1 and ZEN, achieving a degradation rate of 98.91% for AFB1 and 97.69% for ZEN at 50℃. Compared to wild-type BVDyp, mutant M3 maintains good catalytic activity while improving thermal stability and catalytic efficiency. At 50℃, the half-life of M3 increases from 6.87 h for the wild type to 14.73 h, which is 2.14 times that of the wild type. These results indicate that this mutant significantly improves thermal stability.

[0038] 2. UPLC-MS / MS and zebrafish model validation showed that the main degradation products of AFB1 and ZEN were AFQ1 and 15-OH-ZEN, respectively, with lower toxicity than their corresponding protoxins. Mutant M3 still exhibited high detoxification efficiency in actual food or feed matrices, achieving a 66.81% degradation rate of AFB1 in peanut flour and a 63.39% degradation rate of ZEN in corn flour. Compared with other mutants, M3 achieved a better balance between thermal stability and catalytic efficiency, demonstrating the synergistic optimization effect of multi-site combined mutations. Attached Figure Description

[0039] 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.

[0040] Figure 1 The design flowchart (A) and SDS-PAGE protein expression diagram for BVDyp (B).

[0041] Figure 2 Figure 1 shows the enzymatic properties of BVDyp and its degradation characteristics with AFB1 and ZEN. Figure A shows the effect of different temperatures on the degradation of AFB1 and ZEN by BVDyp; Figure B shows the effect of different pH values ​​on the degradation of AFB1 and ZEN by BVDyp; Figure C shows the effect of H2O2 concentration on the degradation of AFB1 and ZEN by BVDyp; Figure D shows the effect of BVDyp enzyme concentration on the degradation of AFB1 and ZEN; Figure E shows the effect of reaction time on the degradation of AFB1 and ZEN by BVDyp; and Figure F shows the effect of the coexistence of AFB1 and ZEN on the degradation of AFB1 and ZEN by BVDyp.

[0042] Figure 3 Figure A shows the degradation product analysis of BVDyp; Figure B shows the degradation product analysis of AFB1 and the degradation product analysis of ZEN.

[0043] Figure 4 Figure A shows the zebrafish toxicity evaluation results; Figure B shows the zebrafish toxicity evaluation of BVDyp degradation of AFB1, Figure C shows the zebrafish toxicity evaluation of BVDyp degradation of ZEN, Figure D shows the liver fluorescence intensity of different treatments, and Figure D shows the levels of ALT, AST, GSH, CAT and MDA in the degradation product treatment groups.

[0044] Figure 5 This is a comparison of the thermal stability of wild-type BVDyp and its mutants at different temperatures.

[0045] Figure 6 Figure 1 shows the degradation properties of mutant M3 on AFB1 and ZEN. Figure 2 shows the effect of different temperatures on the degradation of AFB1 and ZEN by mutant M3, Figure 3 shows the effect of different pH values ​​on the degradation of AFB1 and ZEN by mutant M3, Figure 4 shows the effect of H2O2 concentration on the degradation of AFB1 and ZEN by mutant M3, Figure 5 shows the effect of enzyme concentration of mutant M3 on the degradation of AFB1 and ZEN, Figure 6 shows the effect of reaction time on the degradation of AFB1 and ZEN by mutant M3, and Figure 7 shows the effect of coexistence of AFB1 and ZEN on the degradation of AFB1 and ZEN by mutant M3.

[0046] Figure 7 This study demonstrates the practical application of mutant M3 in the detoxification of peanut flour and corn flour. Detailed Implementation

[0047] 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.

[0048] 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.

[0049] With the development of computational biology and protein engineering technologies, rational design methods based on protein structure prediction, free energy change assessment, and sequence conservation analysis have provided new technical pathways for the targeted optimization of enzyme molecules. In particular, combining protein structure prediction, mutation stability scoring, and sequence evolution information can screen for potential favorable mutation sites in a relatively short time, thereby improving enzyme stability, activity, or substrate adaptability. Compared with random mutagenesis and large-scale high-throughput screening, this strategy has the advantages of strong targeting and high screening efficiency. However, research on targeted thermostability modification of BVDyp, which can simultaneously degrade AFB1 and ZEN, and verification of its detoxification effect in actual food or feed matrices, is still relatively insufficient. Therefore, developing a BVDyp mutant with improved thermostability while maintaining good catalytic performance and applying it to AFB1 and ZEN detoxification has significant application value.

[0050] Example 1: Acquisition and expression of wild-type BVDyp

[0051] 1. Obtaining the BVDyp gene

[0052] Wild-type BVDyp gene origin: The BVDyp encoding gene was obtained by BLASTp alignment of the whole genome sequencing results of the reported DyP-type peroxidase WP_003222196.1 with those of the fungal toxin degrading bacterium HNGD-JQ06 obtained through laboratory screening in the NCBI database. The similarity between this gene and the query sequence was 81.82%.

[0053] 2. Preparation of recombinant Escherichia coli BL21(DE3)

[0054] The BVDyp coding gene with the nucleotide sequence shown in SEQ ID NO.4 was synthesized by Sangon Biotech (Shanghai) Co., Ltd., and optimized according to the codon preference of Escherichia coli BL21(DE3). The optimized sequence is shown in SEQ ID NO.5. The restriction enzyme site Sac1 was introduced at the 5′ end of the coding gene and the restriction enzyme site NotI was introduced at the 3′ end.

[0055] The amino acid sequence of this gene is shown in SEQ ID NO.3. The optimized BVDyp encoding gene was ligated to the corresponding site in the *E. coli* expression vector pET-28a(+) to obtain the recombinant plasmid pET-28a(+)-BVDyp( Figure 1A). The recombinant plasmid was transformed into E. coli DH5α competent cells. After screening and verifying positive clones, the plasmid was extracted and transformed into E. coli expression strain BL21(DE3). Positive clones were screened by LB plates containing kanamycin and verified by bacterial PCR to obtain recombinant E. coli that could express BVDyp.

[0056] 3. Induction and purification of peroxidase BVDyp

[0057] Escherichia coli BL21(DE3) / pET-28a(+)-BVDyp was inoculated into 25 mL of LB medium and cultured at 37°C and 200 rpm with shaking for 12 h. Then, 2% of the culture was inoculated into 300 mL of LB liquid medium and cultured at 37°C and 200 rpm until OD (digesterone) was reached. 600 The concentration was 0.6~0.8, and IPTG was added to a final concentration of 0.1 mmol / L. The mixture was induced at 16℃ for 20 h.

[0058] The induced bacterial culture was collected and centrifuged at 8000 rpm for 10 min at 4℃, and the supernatant was discarded. The bacterial cells were resuspended in 10 mL of equilibration buffer and sonicated for 30 min under ice bath conditions. The sonication power was 300 W, with sonication on for 4 s and off for 4 s. The lysate was centrifuged at 12000 rpm for 10 min at 4℃, and the supernatant was collected and filtered through a 0.45 μm aqueous filter membrane. BVDyp was purified using a Ni-NTA affinity chromatography column, and the target protein was eluted with elution buffer containing 200 mmol / L imidazole. The elution buffer was concentrated and desalted using a 10 kDa ultrafiltration tube to obtain the purified protein. The protein purity was determined by SDS-PAGE, and the results are shown below. Figure 1 As shown in Figure B, the target protein shows a distinct band at approximately 45 kDa, indicating that BVDyp was successfully expressed.

[0059] Example 2: Degradation experiments of AFB1 and ZEN in BVDyp

[0060] The effect of different H2O2 concentrations on the degradation of AFB1 and ZEN by BVDyp was investigated in a 500 μL reaction system. The BVDyp concentration in the reaction system was 20 μg / mL, and the degradation effect was measured after reacting at pH 5 and 40℃ for 24 h.

[0061] The optimal pH for BVDyp degradation of AFB1 and ZEN was determined by conducting the reaction within the pH range of 3-7; the optimal reaction temperature was determined by incubating the reaction system at 30-80℃ for 24 h.

[0062] To further characterize the degradation properties of BVDyp, the effects of different enzyme concentrations and reaction times on the degradation activities of AFB1 and ZEN were investigated.

[0063] In addition, under co-contamination conditions of AFB1 and ZEN, the simultaneous degradation capacity of the two mycotoxins was determined by using 20 μg / mL BVDyp and 0.1 mmol / L H2O2 at 50℃ and pH 6 for 24 h.

[0064] The results are as follows Figure 2 As shown, the BVDyp-H2O2 system provided by this invention can effectively degrade AFB1 and ZEN. At a preferred H2O2 concentration of 0.1 mmol / L, BVDyp exhibits high degradation capacity for both AFB1 and ZEN. Figure 2 C). The optimal pH for BVDyp to degrade AFB1 is 5.0, with a degradation rate of up to 98.91%; the optimal pH for degrading ZEN is 6.0, with a degradation rate of up to 97.69%. Figure 2 B). This system maintains high degradation activity against both toxins within the 30-40℃ range. Figure 2 A). With increasing BVDyp concentration, the degradation rates of AFB1 and ZEN gradually increased. When the enzyme concentration reached 20 μg / mL, the degradation rates of both toxins remained at a high level, and further increasing the enzyme concentration had limited effect on improving the degradation efficiency. Figure 2 D).

[0065] When the BVDyp concentration was 20 μg / mL and the reaction time was 12 h, the degradation rates of AFB1 and ZEN were 77.61% and 73.09%, respectively. After 24 h of reaction, the degradation rates of the two toxins reached 90.54% and 88.94%, respectively. Figure 2 E). In the coexistence system of AFB1 and ZEN, when the concentration of AFB1 was 5 μg / mL and the concentration of ZEN was 10 μg / mL, the degradation rates of AFB1 and ZEN reached 61.68% and 55.83%, respectively, after 24 h of reaction. Figure 2 F).

[0066] Example 3: Safety evaluation of degradation products

[0067] UPLC-MS / MS was used to analyze the products of BVDyp degradation of AFB1 and ZEN, and a zebrafish model was used to evaluate the toxicity differences between the protoxin and the degradation products. The results showed that AFB1, after being treated with BVDyp, mainly generated AFQ1 (…). Figure 3 A), ZEN, after being acted upon by BVDyp, mainly generates 15-OH-ZEN (A). Figure 3 B), the toxicity of both degradation products is lower than that of their corresponding original toxins.

[0068] Safety evaluation results as follows Figure 4As shown, AFB1 treatment significantly reduced the fluorescent area of ​​the liver in zebrafish juveniles, and ZEN treatment caused developmental abnormalities such as spinal curvature and yolk sac edema; while the group treated with products degraded by BVDyp did not show obvious morphological abnormalities, and the liver fluorescent area and intensity were close to those of the normal control group. Figure 4 AC). Furthermore, compared to the protoxin-treated group, the degradation product-treated group showed decreased levels of ALT, AST, and MDA, while CAT and GSH levels recovered. Figure 4 (DH). This indicates that BVDyp significantly reduces the hepatotoxicity, developmental toxicity, and oxidative stress damage of AFB1 and ZEN after degradation.

[0069] Example 4: Rational Design and Construction of BVDyp Mutants

[0070] The three-dimensional structure of BVDyp was predicted using AlphaFold3. Subsequently, structure-energy optimization and mutation energy calculations were performed using FoldX software. First, the RepairPDB module was used to repair and minimize the energy of the AlphaFold3 predicted structure to eliminate unreasonable conformations. Then, the BuildModel module was used to perform virtual saturation mutations on the entire sequence, and the stability change ΔΔG of each mutant relative to the wild type was calculated. In the initial screening, mutants with ΔΔG below -2 kcal / mol were selected as candidate mutants.

[0071] To further screen for biologically plausible mutation sites, a position-specific substitution matrix (PSSM) was introduced as an evolutionary screening criterion. Mutations that simultaneously satisfied a PSSM of -1 and a ΔΔG of -2 kcal / mol were retained, resulting in 23 candidate mutations potentially associated with improved BVDyp thermal stability. Based on structural stability predictions and the spatial relationship between the mutation sites and heme binding and catalytic centers, three mutation sites beneficial to improving BVDyp thermal stability were selected, and combinatorial mutants were constructed based on single-site mutants.

[0072] Specifically, the three preferred mutation sites were designated as mutation site A, mutation site B, and mutation site C. Any two mutation sites were combined to construct three double mutants: A / B, A / C, and B / C, respectively; all three mutation sites were combined simultaneously to construct the A / B / C triple mutant M3. Using the BVDyp encoding gene as a template, primers containing the target mutation sites were designed, and the corresponding mutations were introduced via PCR amplification. The cells were then transformed into host cells, positive clones were screened, and the correct mutation sites were confirmed by sequencing. After sequencing verification, corresponding single-site, double-site, and triple-site mutants were obtained for subsequent expression, purification, and enzymatic property analysis.

[0073] Example 5: Determination of the thermal stability of the mutant

[0074] Three sites showing good thermostability enhancement were screened from candidate mutations, and pairwise and three-site combination mutants were further constructed. Among them, M1 (T46P), M2 (A82L / C405I), and M3 (T46P / A82L / C405I) showed the most significant thermostability improvement. Wild-type BVDyp and each mutant were incubated at 20-80℃ for 3 h, and the residual enzyme activity was measured using ABTS as a substrate. Residual enzyme activity was also measured at different time points after incubation at 50℃, and the half-life was calculated based on thermodynamics.

[0075] Table 1 Half-life of BVDyp and its mutants

[0076]

[0077] Thermal stability test results show that ( Figure 5 As the treatment temperature increased, the residual enzyme activity of both wild-type BVDyp and its mutants gradually decreased, but the decrease in enzyme activity of the mutants was less than that of the wild-type. After treatment at 50℃, the relative enzyme activities of wild-type, M1, M2, and M3 were 36.0±2.0%, 51.10±1.2%, 52.7±1.2%, and 77.10±2.0%, respectively; after treatment at 60℃, the relative enzyme activities of M1, M2, and M3 increased by 18.2±1.0%, 20.5±2.0%, and 48.5±1.2%, respectively, compared with the wild-type. The half-life of M3 at 50℃ reached 14.73 h, while the half-life of wild-type BVDyp was 6.87 h, which was 2.14 times that of the wild-type (Table 1). These results indicate that M3 has higher thermal stability.

[0078] Example 6: Study on the degradation properties of AFB1 and ZEN by the M3 mutant

[0079] Figure 6 This diagram illustrates the degradation properties of AFB1 and ZEN by mutant M3. After mutation, Figure 6 The trend of the effect of H2O2 concentration in C on the degradation rate is basically consistent with that before the mutation. The degradation effect is better in the range of 0.10-0.20 mmol / mL. Among them, the degradation rate of AFB1 is slightly improved overall, while ZEN does not change significantly, indicating that the mutation did not significantly change its optimal requirement for H2O2. Figure 6 In F, the degradation rates of both toxins increased with increasing reaction time when they coexisted. 24 h after the mutation, the degradation rates of AFB1 and ZEN were approximately 66% and 62%, respectively, higher than the pre-mutation rates of approximately 61% and 56%, indicating that the mutation enhanced the enzyme's ability to degrade the coexisting AFB1 and ZEN to some extent.

[0080] M3 can degrade AFB1 and ZEN within the temperature range of 20-80℃, with degradation rates of 98.91% and 97.69% for the two toxins, respectively, at 50℃. Figure 6 A). The optimal pH for M3 degradation of AFB1 is 5.0, with a degradation rate of 96.03%; the optimal pH for ZEN degradation is 6.0, with a degradation rate of 95.87%. Figure 6 B). After 6 h of reaction, the degradation rates of AFB1 and ZEN were 94% and 91%, respectively; after 24 h of reaction, the degradation rates of both toxins were close to 100%. Figure 6 E). Within the enzyme concentration range of 10-50 μg / mL, M3 maintains a high degradation capacity for both AFB1 and ZEN. Figure 6 D).

[0081] Application example: Detoxification effect in actual matrices

[0082] The degradation effects of M3 on AFB1 and ZEN were further evaluated in food matrices. Peanut flour and corn flour were used as food matrices, respectively. AFB1 or ZEN was added, followed by the addition of 20 μg / mL M3 enzyme solution and 0.1 mmol / L H2O2 for reaction. Specifically, after mixing peanut flour or corn flour with water and sterilizing, AFB1 at a final concentration of 5 μg / mL or ZEN at a final concentration of 10 μg / mL was added to a single toxin system. After incubation, the reaction mixture was sonicated and centrifuged, and the supernatant was collected. Residual toxins were extracted with 50 mL of dichloromethane, dried under nitrogen, redissolved in 1 mL of methanol, and detected by HPLC. All experiments were performed in triplicate. The results showed that M3 achieved a degradation rate of 66.81% for AFB1 in peanut flour and a degradation rate of 63.39% for ZEN in corn flour. Figure 7 ).

[0083] In summary, compared with other mutants, M3 exhibits a significant improvement in thermal stability, demonstrating the synergistic optimization effect of multi-site combined mutations. The mutants of this invention can be used to improve the application performance of BVDyp in the detoxification of mycotoxins in food and feed.

[0084] 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 peroxidase mutant M3 with improved thermal stability, characterized in that: The amino acid sequence of the mutant is shown in SEQ ID NO.

1.

2. The encoding gene of the peroxidase mutant M3 according to claim 1, characterized in that: The nucleotide sequence of the encoding gene is shown in SEQ ID NO.

2.

3. A recombinant vector expressing the peroxidase mutant M3 of claim 1, characterized in that: It contains the encoding gene as described in claim 2.

4. A recombinant bacterial strain expressing the peroxidase mutant M3 of claim 1, characterized in that: It contains the coding gene as described in claim 2 or the recombinant vector as described in claim 3.

5. The application of the peroxidase mutant M3 according to claim 1, characterized in that, The application includes any of the following: (1) Application in the degradation of mycotoxins; (2) Application in the preparation of fungal toxin detoxification agents; (3) Application in detoxification of food, feed or agricultural products; (4) Application in improving the thermal stability of peroxidase.

6. The application according to claim 5, characterized in that: The mycotoxin is AFB1 and / or ZEN, wherein the degradation product of AFB1 is AFQ1, and the degradation product of ZEN is 15-OH-ZEN.

7. A method for improving the thermal stability of peroxidase, characterized in that: Using wild-type peroxidase BVDyp with the amino acid sequence shown in SEQ ID NO.3 as the parent, site-directed mutagenesis was performed. The mutations included at least one of mutating threonine at position 46 to proline, alanine at position 82 to leucine, and cysteine ​​at position 405 to isoleucine, to obtain a peroxidase with improved thermostability.

8. A method for removing mycotoxins from a food matrix, characterized in that: The peroxidase mutant M3 described in claim 1 is mixed with the food matrix to be treated, and then H2O2 is added to carry out a detoxification reaction.

9. The method for removing mycotoxins from a food matrix according to claim 8, characterized in that: The food matrix is ​​peanut flour or corn flour, and the mycotoxin is AFB1 and / or ZEN.

10. The method for removing mycotoxins from a food matrix according to claim 9, characterized in that: The peroxidase mutant M3 was used at a concentration of 20-30 μg / mL, and the final concentration of H2O2 was 0.1 mmol / L.

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