Aflatoxin degrading enzyme mutant and application thereof

Abstract

The invention discloses an aflatoxin degrading enzyme mutant and application thereof, and belongs to the technical field of enzyme engineering and microorganisms. In order to solve the technical problems that an existing aflatoxin B1 degrading enzyme mostly depends on a mild operation environment, the degradation performance is poor in a high-temperature environment or an extreme acid-base condition, the application range of the aflatoxin B1 degrading enzyme is remarkably limited and the like, the invention provides an aflatoxin B1 degrading enzyme mutant which can efficiently and rapidly degrade aflatoxin B1, and the aflatoxin B1 degrading enzyme mutant has the advantages that the aflatoxin B1 degrading enzyme mutant has a good application prospect. And the strain also has the characteristics of acid and alkali resistance and high temperature resistance, shows excellent performance in the aspect of aflatoxin B1 pollution treatment, and has a good industrial application prospect. Besides, food-grade safe strains such as saccharomyces cerevisiae, bacillus licheniformis or bacillus subtilis are used as host strains for preparing the aflatoxin B1 degrading enzyme mutant, the degrading effect can be guaranteed, meanwhile, the higher requirement for the safety of enzyme preparations in the feed field can be met, and the application of the aflatoxin B1 degrading enzyme mutant has more advantages.

Description

Technical Field

[0001] This invention belongs to the field of enzyme engineering and microbial technology, specifically relating to an aflatoxin-degrading enzyme mutant and its application. Background Technology

[0002] Mycotoxins are toxic secondary metabolites produced by molds. Common types include aflatoxin (AF), trichothecene toxins (such as T-2 toxin and DON), zearalenone (ZEN), ochratoxin A (OTA), and fumonisin. This type of contamination can occur throughout the entire supply chain of grains and other raw materials, from planting and transportation to processing and storage. When environmental temperature and humidity are suitable, molds proliferate continuously, leading to toxin accumulation and causing grain contamination, which in turn causes significant economic losses to the food industry and livestock farming.

[0003] Among numerous mycotoxins, aflatoxins are the most toxic, especially aflatoxin B1 (AFB1), which is the most widespread and potent contaminant. AFB1 is primarily produced by Aspergillus flavus and Aspergillus parasiticus, and possesses immunosuppressive, mutagenic, and strong carcinogenic properties. It mainly damages the liver and kidneys, and long-term exposure can lead to chronic poisoning and even liver cancer. AFB1 is physically and chemically stable and highly heat-resistant, requiring temperatures above 280°C to decompose; conventional cooking methods cannot destroy it. Furthermore, after animals ingest contaminated feed, the toxins and their metabolites can remain in animal-derived foods such as eggs and dairy products, thus threatening human health through the food chain.

[0004] Currently, methods for degrading aflatoxin mainly include physical, chemical, and biological methods. Physical methods (such as adsorption and heating) easily lead to the loss of nutrients and may introduce contaminants; chemical methods (using chemical reagents to destroy the toxin structure) are often accompanied by the generation of byproducts, which may affect the sensory quality of food and pose potential safety risks. In contrast, biological methods, especially bioenzymatic degradation technology, have become a research hotspot due to their mild operating conditions, high specificity, high safety, and the ease of application of enzyme preparations. The core of this method is to utilize enzymes derived from microorganisms to specifically destroy the molecular structure of the toxin, converting it into low-toxicity or non-toxic products.

[0005] However, the enzymatic method still faces many challenges in practical applications: First, there is a shortage of highly efficient degrading enzymes: the types of highly efficient AFB1 degrading enzymes suitable for industrial production are limited, and most enzymes are difficult to mass-produce; Second, there is insufficient stability and applicability: enzyme activity is easily affected by environmental factors (such as temperature and pH), and some enzymes have poor thermal stability and acid-base tolerance, resulting in unstable degradation effects; Third, there are safety and cost issues: some enzyme-producing strains are pathogenic, posing biosafety risks; at the same time, the enzyme extraction and purification processes are complex, and the production cost is high.

[0006] In summary, aflatoxin (especially AFB1) contamination poses a serious threat to food security and the development of the livestock industry. Existing physical and chemical methods have significant limitations, while the more promising enzymatic method is constrained by bottlenecks such as insufficient enzyme resources, poor stability, and high costs. Therefore, to effectively control AFB1 contamination, it is urgent to further explore new safe and efficient degradation enzyme resources to enrich the enzyme library; to thoroughly elucidate the enzyme's mechanism of action and optimize production processes to improve stability and reduce costs; and to strengthen the safety evaluation and compliance research of enzyme preparations to promote their application in actual production, ultimately ensuring food safety and human health. Summary of the Invention

[0007] To address the technical problems of existing aflatoxin B1 degrading enzymes, which rely heavily on mild operating environments and exhibit poor degradation performance under high-temperature or extreme acid / alkali conditions, significantly limiting their application, this invention provides an aflatoxin B1 degrading enzyme mutant, along with the gene encoding this mutant, a recombinant vector carrying the gene, and recombinant bacteria. Furthermore, this invention provides a specific preparation method for the aflatoxin B1 degrading enzyme mutant and clarifies its application effect in aflatoxin B1 degradation. Experimental verification shows that when this mutant is used for aflatoxin B1 degradation, it can stably exert its degradation activity under reaction conditions of 20-90℃ and pH 2-9. This enzyme does not require a mild operating environment and can be directly used as a feed additive, showing broad application prospects in the field of aflatoxin B1 contamination control and playing a significant role in ensuring food and feed safety.

[0008] To solve the above-mentioned technical problems and achieve the corresponding technical effects, the present invention provides the following technical solution: The first objective of this invention is to provide an aflatoxin B1 degrading enzyme mutant, the amino acid sequence of which is shown in SEQ ID NO.1.

[0009] The aflatoxin B1 degrading enzyme mutant was obtained by using the wild-type aflatoxin B1 degrading enzyme with the amino acid sequence shown in SEQ ID NO.2 as the parent, and by mutating aspartic acid at position 14 to arginine, glycine at position 209 to proline, aspartic acid at position 432 to lysine, and glycine at position 672 to proline; the encoding gene of the wild-type aflatoxin B1 degrading enzyme is shown in SEQ ID NO.4.

[0010] A second objective of this invention is to provide a gene encoding the aforementioned aflatoxin B1 degrading enzyme mutant, the nucleotide sequence of which is shown in SEQ ID NO.3.

[0011] A third objective of the present invention is to provide a recombinant vector containing the aforementioned genes.

[0012] In one embodiment of the present invention, the plasmids used in constructing the above-mentioned recombinant vector are pET24a, pHY300PLK, pBE-S, pub110, YEp352 or pPIC9.

[0013] A fourth objective of this invention is to provide a recombinant bacterium containing the aforementioned gene or the aforementioned recombinant vector.

[0014] In one embodiment of the present invention, the host bacterium selected for constructing the above-mentioned recombinant bacteria is *Escherichia coli* (E. coli). Escherichia coli Bacillus subtilis ( Bacillus subtilis ), Bacillus licheniformis ( Bacillus lincheniform Pichia pastoris () Shepherd's pie ) or brewer's yeast ( Saccharomyces yeast ).

[0015] A fifth object of the present invention is to provide an additive containing the aforementioned aflatoxin B1 degrading enzyme mutant.

[0016] In one embodiment of the present invention, the core components of the additive include the above-mentioned aflatoxin B1 degrading enzyme mutant and / or the beneficial substances produced during the fermentation process of the above-mentioned recombinant bacteria. The additive can be used to promote the degradation of aflatoxin B1 and improve feed safety.

[0017] A sixth object of the present invention is to provide a feed containing the above-mentioned aflatoxin B1 degrading enzyme mutant or the above-mentioned additives.

[0018] The seventh objective of this invention is to provide a method for preparing the above-mentioned aflatoxin B1 degrading enzyme mutant, the method comprising the following steps: constructing a recombinant expression vector containing the above-mentioned gene, introducing the recombinant expression vector into a host bacterium to obtain recombinant bacteria; culturing the recombinant bacteria in a liquid culture medium to obtain a bacterial solution, centrifuging to obtain bacterial cells; sonicating and centrifuging, collecting the supernatant as a crude enzyme solution; and performing affinity chromatography on a Ni column to obtain a purified enzyme solution.

[0019] An eighth object of the present invention is to provide the application of the above-mentioned aflatoxin B1 degrading enzyme mutant, the above-mentioned gene, the above-mentioned recombinant vector, the above-mentioned recombinant bacteria or the above-mentioned additive, wherein the application is for degrading aflatoxin B1.

[0020] In one embodiment of the present invention, the application is to degrade aflatoxin B1 in feed.

[0021] The ninth objective of this invention is to provide a method for degrading aflatoxin B1, wherein the above-mentioned aflatoxin B1 degrading enzyme mutant or the above-mentioned additive is added to a sample to be treated containing aflatoxin B1, and enzymatically hydrolyzed for 6-96 h at a reaction condition of 20-90°C and pH 2-9.

[0022] In one embodiment of the present invention, the reaction conditions are enzymatic hydrolysis for 24-96 h at 30-70°C and pH 4-9.

[0023] Preferably, the reaction conditions are enzymatic hydrolysis at 30-60°C and pH 6-8 for 36-60 h.

[0024] The beneficial effects of this invention are: The aflatoxin B1 degrading enzyme mutant provided by this invention enriches the types of aflatoxin B1 degrading enzymes; the mutant has excellent temperature and acid and alkali resistance (it can stably exert its activity under conditions of 20-90℃ and pH 2-9), which solves the technical problems of existing biodegradation technologies that rely on mild operating environments and have limited application scenarios, and provides possibilities for diversified scenarios (such as feed processing and agricultural product storage).

[0025] This invention breaks through the traditional limitation of Escherichia coli to a single host. It can use food-grade safe strains such as Bacillus subtilis, Bacillus licheniformis, and Saccharomyces cerevisiae as host bacteria to prepare the aflatoxin B1 degrading enzyme mutant provided by this invention. The produced degrading enzyme has no safety risks and is more suitable for applications in the feed and food industries. At the same time, the aflatoxin B1 degrading enzyme mutant can be directly added to feed as an additive or degrade aflatoxin B1 in the form of an enzyme preparation, which can effectively protect the quality of agricultural products and feed and protect the health of humans and animals. Attached Figure Description

[0026] Figure 1 The image shows the results of colony PCR verification of the recombinant plasmid pET24a-M; where 1, 2, 3, and 4 are single clones, and M is the marker. Detailed Implementation

[0027] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings. It should be noted that the embodiments mentioned below are only for explaining the invention and are not intended to limit the scope of the invention. The embodiments mentioned below are only some embodiments of the invention, not all embodiments. Those skilled in the art can refer to the content of this document and appropriately improve the process parameters to achieve the objectives of the invention. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in the invention. The methods and applications of this invention have been described through preferred embodiments, and those skilled in the art can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content and scope of this invention to realize and apply the technology of this invention. In the art, embodiments obtained by other those skilled in the art without creative effort are all protected by this invention.

[0028] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, and the materials, reagents, culture media and instruments used are conventional materials, reagents, culture media and instruments in the art, which can be obtained by those skilled in the art through commercial channels.

[0029] The reagents involved in this invention are as follows: Aflatoxin B1 standard reference was purchased from Sigma-Aldrich. Phosphate buffer (PBS buffer): Contains 0.24 g / L potassium dihydrogen phosphate, 1.44 g / L disodium hydrogen phosphate, 8 g / L sodium chloride, 0.2 g / L potassium chloride, with the balance being water, and a pH of 7.2.

[0030] The culture medium involved in this invention is as follows: LB liquid medium (1 L): 10 g sodium chloride, 5 g yeast extract, 10 g peptone, deionized water to a final volume of 1 L, autoclave at 121℃ for 20 minutes before use; LB solid medium is the same as liquid medium with 20 g / L agar added, and sterilized in the same way.

[0031] TB liquid culture medium (1 L): 12 g peptone, 24 g yeast extract, 16.4 g dipotassium hydrogen phosphate trihydrate, 5 g glycerol, 2.3 g potassium dihydrogen phosphate, deionized water to a final volume of 1 L, autoclave at 121℃ for 20 minutes before use.

[0032] YPD liquid medium (1 L): 10 g yeast extract, 20 g peptone, 20 g glucose; YPD solid medium is the liquid medium with 20 g / L agar added.

[0033] The methods for detecting aflatoxin content are as follows: The detection method for aflatoxin was carried out in accordance with GB5009.22-2016 to ensure the accuracy and reliability of the test results. Degradation efficiency was assessed using a quantitative formula to calculate the degradation rate: Degradation rate (%) = (Total aflatoxin in the sample before reaction - Mass of residual aflatoxin in the sample after reaction) / Total aflatoxin in the sample before reaction × 100%. This indicator directly reflects the enzyme preparation's ability to degrade aflatoxin.

[0034] Example 1: Preparation of aflatoxin B1 degrading enzyme mutants using an Escherichia coli expression system and determination of their activity. The amino acid sequence of the aflatoxin B1 degrading enzyme mutant prepared in this embodiment is shown in SEQ ID NO.1. This aflatoxin B1 degrading enzyme mutant was obtained by mutation of the wild-type aflatoxin B1 degrading enzyme with the amino acid sequence shown in SEQ ID NO.2.

[0035] SEQ ID NO.1 (D14R / G209P / D432K / G672P): MATTTVHRERFLA R KSAPLCGMDIRKSFDQLSSKEKLYTHYVTEASWAGARIIQAQWTPQATDLYDLLILTFSVNGKLADLNALKTSSGLSEDDWEALIQYTVQVLSNLVNYKTFGFTKIIPRVDAEKFESVVKASSNADQGSALFTKLKQHIYALSPESALFIGKRKDGHVSNYYLGEPVGDAEVDAIQNVAEKLGVDILNTRVKKN P AGDYTLLVASAKTSPPSVHDFQIDSTPAKLTIEYGDYASSLTKVVAALQEAKQYTANDHQSAMIEGYVKSFNSGSIPEHKAASTEWVKDIGPVVESYIGFVETYVDPYGGR AEWEGFTAIVDKQLSAKYEALVNGAPKLIKSLPWGTDFEVDVFRKPDFTALEVVSFATGGIPAGINIPNYYEVRESTGFKNVSLANILAAKVPNEELTFIHPDDVELYNAW KSRAFELQVANHELLGHGSGKLFQEGADGKLNFDPEKVINPLTGKPITSWYKPGQTPDSVLGEVSSSMEECRAETVALYLVSNLDILKIFNYVDKQDIEDIQYITFLLMARAGLRALEFY DPATKKHGQAHMQARMGITQYLIQAGIARLELIQDANGELENLYVRVDREKVLSKGKEVVGQLLIELQVRKSTADGTGSRDFYTTLTEPISGWEGKIRDIVLKKKLPRKIFVQPNTFVVN P EVQLKEYPLTAAGVIESFIERRL.

[0036] 1. Obtaining the gene fragment encoding the aflatoxin B1 degrading enzyme mutant A nucleotide sequence encoding the aflatoxin B1 degrading enzyme mutant shown in SEQ ID NO.3 was added to the 5' end. BamH I restriction site (GGATCC), added at the 3' end Salt The above gene fragment was synthesized by Genewiz Inc. using artificial chemical methods for the I restriction site (GTCGAC).

[0037] SEQ ID NO.3 (underlined area indicates mutation site): ATGGCCACCACAACTGTCCACCGGGAGCGATTCCTGGCA CGTAAGTCTGCTCCTTTGTGTGGTATGGATATTAGAAAGTCATTTGATCAGCTCAGCTCTAAGGAAAAGCTCTACACGCATTACGTGACCGAAGCTTCTTGGGCGGGCGCAAGAATCATCCAGGCTCAGTGGACCCCGCAGGCGACAGATCTATATGATCTGTTGATCCTTACGTTCAGCGTAAATGGAAAGCTCGCCGACCTGAATGCCCTTAAGACGTCGTCAGGCCTTTCAGAGGACGATTGGGAGGCCTTGATACAGTACACGGTCCAGGTATTGAGCAATCTTGTCAACTACAAGACGTTCGGATTTACGAAGATCATTCCCCGCGTCGACGCAGAAAAGTTTGAGTCAGTGGTCAAAGCCTCTAGCAACGCAGACCAGGGCTCGGCACTATTCACCAAGTTGAAACAACACATATATGCGCTTTCTCCTGAGTCAGCGCTATTCATTGGCAAAAGGAAGGACGGTCACGTATCAAATTACTATCTTGGTGAACCTGTTGGAGATGCTGAGGTCGATGCTATCCAGAATGTCGCTGAGAAGTTAGGCGTTGATATCCTCAATACTCGCGTGAAGAAGAAT 100GCGGGTGATTACACGCTCTTAGTTGCCTCTGCTAAAACCAGTCCACCCTCCGTGCATGACTTCCAAATCGACTCAACTCCGGCTAAATTGACGATTGAGTATGGCGACTACGCGTCATCTCTAACGAAGGTTGTCGCCGCCCTTCAGGAGGCCAAACAGTATACCGCGAACGATCATCAATCAGCGATGATCGAAGGCTATGTCAAGTCGTTCAACTCAGGATCAATTCCGGAACACAAAGCTGCGTCAACAGAATGGGTGAAAGATATTGGACCGGTTGTAGAGTCCTACATCGGGTTCGTCGAAACCTATGTCGACCCATATGGCGGACGCGCGGAATGGGAGGGTTTCACTGCCATCGTCGACAAGCAGCTGAGTGCGAAGTACGAAGCATTGGTTAACGGTGCTCCTAAGTTGATCAAGAGTCTTCCGTGGGGAACGGACTTCGAGGTTGACGTCTTCAGGAAGCCGGACTTTACTGCGTTGGAAGTCGTATCATTTGCAACAGGAGGTATTCCTGCCGGAATCAATATACCAAACTATTATGAAGTCCGGGAAAGCACAGGGTTTAAGAATGTTTCGCTAGCGAATATTTTGGCGGCCAAGGTACCAAACGAGGAGTTAACTTTCATCCATCCTGATGACGTAGAACTATATAACGCTTGG AAGAGTCGCGCGTTTGAACTTCAGGTGGCCAACCACGAACTTTTGGGTCATGGCTCCGGCAAGCTTTTCCAAGAAGGTGCTGATGGGAAACTGAACTTCGATCCCGAAAAGGTCATAAACCCTCTGACTGGAAAGCCGATAACTTCATGGTATAAGCCAGGGGCAAACGCCGGATTCTGTTTT AGGCGAAGTGTCGTCGTCAATGGAAGAATGTCGGGCGGAGACCGTAGCGCTCTACTTGGTTAGCAACCTCGATATTTCTAAAATTTTCAATTACGTCGACAAGCAAGACATTGAAGATATCCAGTACATCACGTTCTTGCTTATGGCCCGCGCTGGTCTGCGGGCACTAGAGTTTTATG ATCCAGCCACCAAGAAGCACGGACAGGCACATATGCAGGCCAGAATGGGCATAACCCAGTACCTGATTCAAGCTGGGATTGCGAGACTTGAATTGATCCAGGATGCCAACGGCGAACTCGAAAACTTATACGTTCGGGTTGACCGGGAGAAAGTGTTGTCCAAAGGAAAGGAGGTTGTT GGTCAATTGCTGATCGAACTCCAAGTCCGGAAAAGTACCGCAGACGGCACCGGCTCCCGAGATTTCTACACAACGCTGACCGAACCAATCTCTGGATGGGAGGGCAAGATCCGAGACATCGTTTTGAAGAAGAAGCTTCCTCGAAAAAATCTTTGTCCAACCCAATACATTTGTCGTCAAC 300 GAAGTCCAGCTCAAAGAGTATCCTTTGACGGCTGCCGGGGTAATTGAAAGTTTCATTGAGAGACGATTGTGA.

[0038] 2. Construction of recombinant plasmids 1) Use Takara restriction endonucleases BamH I and Salt I. The gene fragment obtained in step 1 and the pET22b plasmid were double-digested with enzymes. The enzyme digestion reaction system for the gene fragment is as follows: 1 µL Salt I, 1 µL BamHI. 4.5 µL 1.5T×buffer, 10 µL gene fragment (96 ng / µL), 13.5 µL deionized water; reaction conditions: 37℃ for 2 h. After the enzyme digestion reaction, nucleic acid gel recovery was performed to obtain the gene fragment (75 ng / µL). The enzyme digestion reaction system for pET22b plasmid is as follows: 1 µL BamH I, 1 µL Salt I. 4.5 µL 1.5 T×buffer, 10 µL pET22b (101 ng / µL), 13.5 µL deionized water; reaction conditions were 37℃ for 2 h. After the enzyme digestion reaction, nucleic acid gel recovery was performed to obtain the linear plasmid pET24a fragment (83 ng / µL).

[0039] 2) The double-digested gene fragment was ligated with the double-digested plasmid pET24a using T4 ligase to obtain the ligation product. The specific reaction system was as follows: 1 µL T4 DAN ligase, 1 µL T4 DAN ligase buffer, 2 µL gene fragment (75 ng / µL), 2 µL pET24a fragment (83 ng / µL), and 4 µL deionized water. The ligation reaction was carried out at 16℃ for 4 h.

[0040] 3) Add 10 µL of the ligation product obtained in step 2) to 100 μL of ice-cold *E. coli* DH5α competent cells. After incubating on ice for 30 minutes, heat shock at 42°C for 90 seconds, followed by immediate ice incubation for 2 minutes. Add 1 mL of LB liquid medium and incubate at 37°C with shaking for 45 minutes. Plate the culture and screen by spreading the bacterial culture onto LB agar plates containing a final concentration of 50 μg / mL kanamycin. Incubate overnight at 37°C with the plates inverted. Select 4 single clones for colony PCR verification. The PCR reaction system is as follows: 1 µL of upstream primer (nucleotide sequence as shown in SEQ ID NO.5: ATGGCCACCACAACTGTCCACCG), 1 µL of downstream primer (nucleotide sequence as shown in SEQ ID NO.6: TCACAATCGTCTCTCAATGAAAC), 0.5 μL of colony template, 25 μL of DNA polymerase 2×Prime STAR MaxDNA, and sterile water to a final volume of 50 μL. The PCR reaction procedure is as follows: 98℃ pre-denaturation for 5 minutes; 98℃ denaturation for 30 seconds, 55℃ annealing for 30 seconds, 72℃ extension for 2 minutes, 30 cycles; final extension at 72℃ for 5 minutes. Take 3 μL of the colony PCR product for nucleic acid verification. The target band size is 2088 bp. Figure 1As shown, the bands of single clones 1, 2, 3, and 4 are of the correct size. Select the correctly PCR-positive single clones and culture them in LB medium. Then, extract the plasmid for sequencing to obtain the correct recombinant plasmid, denoted as pET24a-M.

[0041] 3. Obtaining recombinant strains The correctly sequenced recombinant plasmid was transformed into *E. coli* BL21(DE3) cells. The specific procedure was as follows: 5 μL of the recombinant plasmid was added to 100 μL of pre-chilled *E. coli* BL21(DE3) competent cells and incubated on ice for 30 minutes. A heat shock at 42°C for 90 seconds was performed, followed by an immediate ice incubation for 2 minutes. 1 mL of LB medium was added, and the cells were cultured at 37°C with shaking at 200 rpm for 45 minutes. The cells were then plated for screening. The bacterial culture was spread on LB plates containing a final concentration of 50 μg / mL kanamycin and incubated upside down overnight at 37°C. Single clones were selected and cultured in LB medium. The recombinant strain BL21 / pET24a-M was obtained after identification. 4. Enzyme Preparation The recombinant strain BL21 / pET24a-M obtained in step 3 was inoculated into a 100 mL Erlenmeyer flask containing 10 mL of LB liquid medium and activated by incubation at 37°C for 12 h. 1 mL of the activated bacterial culture was transferred to 50 mL of TB liquid medium and incubated at 37°C for 32 h. The bacterial cells were then collected by centrifugation. After resuspending the bacterial cells in phosphate buffer, they were sonicated and centrifuged at 4°C and 10,000 rpm for 20 min. The supernatant was collected as the crude enzyme solution.

[0042] The crude enzyme solution was purified by Ni column affinity chromatography: Since the target recombinant enzyme is fused with a His tag, the Ni column was first equilibrated with a buffer containing 50 mM NaCl and 10 mM imidazole (pH 7.4) at a flow rate of 1 mL / min; then, the crude enzyme solution sample was circulated and loaded at a flow rate of 1 mL / min to ensure that the His-tagged recombinant protein was fully bound; then, it was washed with the above buffer at a flow rate of 1 mL / min to remove non-specifically bound impurity proteins; finally, it was eluted with a buffer containing 50 mM NaCl and 500 mM imidazole (pH 7.4) to collect the high-purity enzyme solution.

[0043] 5. Enzyme activity detection Enzyme activity assay: Take 1000 µL of the 10 mg / mL high-purity enzyme solution obtained in step 4, transfer it to a 1.5 mL centrifuge tube, add aflatoxin B1 standard solution and mix thoroughly to make the final concentration of aflatoxin B1 in the mixture 40 ppm.

[0044] 1) The effect of reaction time on enzyme activity To further investigate the effect of reaction time on enzyme activity, the mixture was incubated at 37℃ and pH 7. 20 µL samples were collected at 6 h, 12 h, 24 h, 36 h, 48 h, 60 h, 72 h, and 96 h to detect aflatoxin B1 residues. Table 1 shows that the degradation efficiency of aflatoxin B1 exceeded 90% after 48 h, and the rate of increase slowed down with further time. Therefore, a reaction time of 50 h was selected for subsequent experiments.

[0045] 2) The effect of temperature on enzyme activity To further investigate the effect of reaction temperature on enzyme activity, eight identical mixed solutions were reacted at pH 7 at 20℃, 30℃, 40℃, 50℃, 60℃, 70℃, 80℃, and 90℃ for 50 h. 20 µL samples were taken from each solution to detect aflatoxin B1 residues. Table 2 shows that the degradation efficiency was highest at 40℃, with good results at 30℃, 50℃, 60℃, and 70℃. The degradation efficiency decreased above 70℃ but remained effective; therefore, 40℃ was selected as the reaction temperature for subsequent experiments.

[0046] 3) The effect of pH on enzyme activity To further investigate the effect of reaction temperature on enzyme activity, eight identical mixed solutions were adjusted to pH values ​​of 2, 3, 4, 5, 6, 7, 8, and 9 under a constant temperature of 40℃. After 50 h of reaction, 20 µL samples were taken from each solution to detect aflatoxin B1 residues. Table 3 shows that the degradation efficiency was highest at pH 7, with good results at pH 5, 6, 8, and 9. The degradation efficiency decreased below pH 4 but remained effective.

[0047] Example 2: Preparation of aflatoxin B1 degrading enzyme mutant using a Bacillus subtilis expression system and determination of its activity. This embodiment aims to illustrate the preparation method and application of aflatoxin B1 degrading enzyme mutant with an amino acid sequence as shown in SEQ ID NO.1, focusing on verifying the effect of the Bacillus subtilis expression system on the enzyme preparation efficiency and activity, and providing a basis for diversified host selection for its industrial production.

[0048] The core difference between the preparation method of the aflatoxin B1 degrading enzyme mutant in this embodiment and that in Example 1 lies in the different selection of the expression vector and the host strain: In step 2, when constructing the recombinant vector, the Bacillus subtilis expression vector pBE-S was selected (this vector has a high-efficiency prokaryotic expression element and is conducive to the secretion of the target protein); in step 3, the recombinant vector was transformed into Bacillus subtilis ( Bacillus subtilis WB600 (this strain lacks the main protease gene, which can reduce the degradation of the target protein and improve expression stability) was finally obtained as a recombinant strain, denoted as WB600 / pBE-SM.

[0049] In subsequent operations, the method for preparing high-purity enzyme solution by fermentation using recombinant strain WB600 / pBE-SM strictly followed the same culture conditions, cell disruption, and purification procedures as in Example 1 to ensure the comparability of experimental data.

[0050] In the enzyme activity evaluation stage, the degradation efficiency of aflatoxin B1 was evaluated by detecting different reaction times (6-96 h), temperatures (20-90℃), and pH (2-9). The relevant results are shown in Table 1 (influence of time), Table 2 (influence of temperature), and Table 3 (influence of pH).

[0051] Example 3: Preparation of aflatoxin B1 degrading enzyme mutant using a Bacillus subtilis expression system and determination of its activity. This embodiment aims to illustrate the preparation method and application of aflatoxin B1 degrading enzyme mutant with an amino acid sequence as shown in SEQ ID NO.1, focusing on verifying the effect of the Bacillus licheniformis expression system on the preparation efficiency and activity of this enzyme, and providing a basis for diversified host selection for its industrial production.

[0052] The core difference between the preparation method of the aflatoxin B1 degrading enzyme mutant in this embodiment and that in Example 1 lies in the different selection of the expression vector and the host strain: In step 2, when constructing the recombinant vector, Bacillus licheniformis (… Bacillus Lichen-shaped The specific expression vector pHY300PLK contains a strong promoter and stable replication element of Bacillus licheniformis, which is adapted to its genetic background to improve expression efficiency. In step 3, the recombinant vector is transformed into Bacillus licheniformis (this strain is widely used in the production of enzyme preparations in the food industry due to its characteristics such as high temperature resistance and strong stress resistance) to obtain the recombinant strain, denoted as BL / pHY300PLK-M.

[0053] In subsequent operations, the method for preparing high-purity enzyme solution by fermentation using recombinant strain BL / pHY300PLK-M strictly followed the same culture conditions, cell disruption and purification procedures as in Example 1 to ensure the comparability of experimental data.

[0054] In the enzyme activity evaluation stage, the degradation efficiency of aflatoxin B1 was evaluated by detecting different reaction times (6-96 h), temperatures (20-90℃), and pH (2-9). The relevant results are shown in Table 1 (influence of time), Table 2 (influence of temperature), and Table 3 (influence of pH).

[0055] Example 4: Preparation of aflatoxin B1 degrading enzyme mutants using a Saccharomyces cerevisiae expression system and determination of their activity. This embodiment aims to illustrate the preparation method and application of aflatoxin B1 degrading enzyme mutant with an amino acid sequence as shown in SEQ ID NO.1, focusing on verifying the impact of eukaryotic expression system on the enzyme preparation efficiency and activity, and providing a basis for diverse host selection for its industrial production.

[0056] The core difference between the preparation method of the aflatoxin B1 degrading enzyme mutant in this embodiment and that in Example 1 lies in the selection of the expression vector and the host strain: In step 2, when constructing the recombinant vector, the yeast expression vector YEp352 was selected. This vector contains a strong promoter of Saccharomyces cerevisiae (such as the ADH1 promoter) and a 2μm origin of replication, which is adapted to the transcriptional regulation mechanism of the eukaryotic host and can efficiently drive the expression of the target gene; in step 3, the recombinant vector is transformed into Saccharomyces cerevisiae (… Saccharomyces yeast This strain possesses natural advantages in the production of food-grade enzymes due to its food-grade safety, well-developed post-translational modification system, and good fermentation compatibility. Furthermore, the strain was cultured using YPD medium adapted to yeast. The resulting recombinant strain was designated SC / YEp352-M.

[0057] In subsequent operations, the method for preparing high-purity enzyme solution using recombinant strain SC / YEp352-M fermentation strictly followed the same culture conditions, cell disruption, and purification procedures as in Example 1 to ensure the comparability of experimental data.

[0058] In the enzyme activity evaluation stage, the degradation efficiency of aflatoxin B1 was evaluated by detecting different reaction times (6-96 h), temperatures (20-90℃), and pH (2-9). The relevant results are shown in Table 1 (influence of time), Table 2 (influence of temperature), and Table 3 (influence of pH).

[0059] Comparative Example 1: Preparation of wild-type aflatoxin B1 degrading enzyme using an Escherichia coli expression system and determination of its activity. The preparation method of the wild-type aflatoxin B1 degrading enzyme (amino acid sequence as shown in SEQ ID NO.2) provided in this comparative example differs from the preparation method of the aflatoxin B1 degrading enzyme mutant described in Example 1 only in that the nucleotide sequence encoding the wild-type aflatoxin B1 degrading enzyme in step 1 is as shown in SEQ ID NO.4. This comparative example uses the same method as Example 1, adding restriction sites to both ends of the nucleotide sequence shown in SEQ ID NO.4 and artificially synthesizing gene fragments. After constructing a recombinant vector and a recombinant strain, a recombinant strain BL21 / pET24a-WT was obtained. Using this strain, a high-purity enzyme solution was prepared according to the same method as in Example 1. The enzyme activity was evaluated by detecting the degradation efficiency of the wild-type aflatoxin B1 degrading enzyme under different reaction times, temperatures, and pH conditions (see the method in Example 1). The degradation rate results for different reaction times are shown in Table 1, the degradation rate results for different reaction temperatures are shown in Table 2, and the degradation rate results for different pH values ​​are shown in Table 3.

[0060] SEQ ID NO.2: ; SEQ ID NO.4:

[0061] Comparative Example 2: Blank Vector Control Experiment (Blank Control) Aflatoxin B1 degrading enzyme was prepared according to the method of Example 1, except that the blank vector pET22b plasmid without gene transfer was used instead of the recombinant plasmid used in Example 1.

[0062] Table 1. Detection results of aflatoxin B1 degradation rate of enzyme solutions obtained in each example at different reaction times.

[0063] Table 2. Detection results of aflatoxin B1 degradation rate of enzyme solutions obtained in each example at different reaction temperatures.

[0064] Table 3. Detection results of the degradation rate of aflatoxin B1 by the enzyme solutions obtained in each example at different pH values.

[0065] As shown in Table 1, under all reaction time conditions, the degradation rate of aflatoxin B1 by the aflatoxin B1 degrading enzyme mutants prepared in Examples 1-4 was significantly higher than that of the wild-type aflatoxin B1 degrading enzyme prepared in Comparative Example 1. When the reaction time was 24 h, the degradation rate of aflatoxin B1 by the aflatoxin B1 degrading enzyme mutants prepared in Examples 1-4 all exceeded 50%, and the degradation rate gradually increased with the extension of the reaction time. When the reaction time was 36 h, the degradation rate of aflatoxin B1 by the aflatoxin B1 degrading enzyme mutants prepared in Examples 1-4 all exceeded 85%.

[0066] As shown in Table 2, under all temperature and time conditions, the aflatoxin B1 degrading enzyme mutants prepared in Examples 1-4 showed significantly higher degradation rates of aflatoxin B1 than the wild-type aflatoxin B1 degrading enzyme prepared in Comparative Example 1. Within the temperature range of 30-70℃, the aflatoxin B1 degrading enzyme mutants prepared in Examples 1-4 showed degradation rates exceeding 65%, and within the temperature range of 30-60℃, the aflatoxin B1 degrading enzyme mutants prepared in Examples 1-4 showed degradation rates close to or exceeding 80%.

[0067] As shown in Table 3, under all pH conditions, the aflatoxin B1 degrading enzyme mutants prepared in Examples 1-4 exhibited significantly higher degradation rates of aflatoxin B1 than the wild-type aflatoxin B1 degrading enzyme prepared in Comparative Example 1. Under pH conditions of 4-9, the aflatoxin B1 degrading enzyme mutants prepared in Examples 1-4 all showed degradation rates exceeding 50%, and under pH conditions of 6-8, the aflatoxin B1 degrading enzyme mutants prepared in Examples 1-4 all showed degradation rates exceeding 90%.

[0068] Example 5: Application of aflatoxin B1 degrading enzyme mutant in the degradation of corn steep liquor containing aflatoxin B1 This embodiment aims to verify the degradation ability of aflatoxin B1 degrading enzyme mutants prepared from different host bacteria, with amino acid sequences as shown in SEQ ID NO.1, and wild-type aflatoxin B1 degrading enzymes prepared from Comparative Example 1, with amino acid sequences as shown in SEQ ID NO.2, on aflatoxin B1 in corn steep liquor, providing data support for the application scenarios of aflatoxin B1 degrading enzyme mutants.

[0069] The experimental procedure was as follows: High-purity enzyme solutions prepared in Examples 1-4 were mixed with corn steep liquor containing 30 ppm aflatoxin B1. The enzyme concentration was precisely adjusted according to the quality of the corn steep liquor to ensure a stable final enzyme concentration of 5 ppm in the mixed system, thus eliminating interference from enzyme concentration differences on the degradation results. The mixed system was then reacted at 40℃ and pH 7 for 50 hours (these conditions were the optimal parameters for enzyme activity determined in previous experiments). The aflatoxin B1 residue was measured to calculate the degradation efficiency.

[0070] The test results are shown in Table 4: the pure enzymes of Example 1 (host: *Escherichia coli*), Example 2 (host: *Bacillus subtilis*), Example 3 (host: *Bacillus licheniformis*), and Example 4 (host: *Saccharomyces cerevisiae*) showed degradation efficiencies of 85.7%, 86.4%, 84.7%, and 84.3% for aflatoxin B1 in corn steep liquor, respectively. It can be seen that although Example 1 used *Escherichia coli* as the host, and Examples 2-4 used food-grade safe strains such as *Bacillus subtilis*, *Bacillus licheniformis*, and *Saccharomyces cerevisiae* as hosts, there was no significant difference in degradation efficiency between the enzymes prepared from both methods. This result indicates that using food-grade safe strains as hosts to produce this degrading enzyme mutant can ensure degradation efficiency while meeting the higher safety requirements for enzyme preparations in the feed industry, making its application more advantageous.

[0071] Furthermore, comparing the degradation effects of Comparative Example 1 (wild-type aflatoxin B1 degrading enzyme) and Example 1 (aflatoxin B1 degrading enzyme mutant): the degradation efficiency of the pure enzyme prepared in Comparative Example 1 was 33.6%, while the degradation efficiency of the pure enzyme prepared in Example 1 was 85.7% (data shown in Table 4). Calculations show that the aflatoxin B1 degrading enzyme mutant of Example 1 has a degradation efficiency 155.06% higher than that of the wild-type enzyme in Comparative Example 1, fully demonstrating that the aflatoxin B1 degrading enzyme mutant with the amino acid sequence shown in SEQ ID NO. 1 significantly enhances the degradation ability of aflatoxin B1 compared to the wild-type enzyme.

[0072] Table 4. Results of the degradation rate of aflatoxin B1 in corn steep liquor by various enzymes after 50 h of reaction at 40℃ and pH 7.

[0073] To verify the degradation performance of the aflatoxin B1 degrading enzyme mutant under extreme conditions, high-purity enzyme solutions of wild-type aflatoxin B1 degrading enzyme (Comparative Example 1) and the aflatoxin B1 degrading enzyme mutant (Example 1) were mixed with corn steep liquor containing 20 ppm aflatoxin B1. The enzyme concentration was precisely controlled according to the quality of the corn steep liquor to ensure that the final enzyme concentration in the mixed system was 5 ppm, so as to eliminate the interference of concentration differences on the results.

[0074] Subsequently, the mixture was reacted at 70℃ and pH 4.0 for 50 h, and the degradation rate of aflatoxin B1 was measured. The results are shown in Table 5: The aflatoxin B1 degradation enzyme mutant enzyme solution prepared in Example 1 showed a 156.7% higher degradation efficiency of aflatoxin B1 compared to the wild-type enzyme solution prepared in Comparative Example 1.

[0075] This result shows that the aflatoxin B1 degrading enzyme mutant provided in Example 1 can still effectively degrade aflatoxin B1 in corn steep liquor under harsh conditions of high temperature (70°C) and low pH (4.0), fully demonstrating its good environmental adaptability and indicating that the enzyme has excellent application potential in industrial production (such as feed processing and other scenarios that require tolerance to extreme process conditions).

[0076] Table 5. Results of the degradation rate of aflatoxin B1 in corn steep liquor by various enzymes at 70℃ and pH 4 for 50 h.

[0077] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A mutant of aflatoxin B1 degrading enzyme, characterized in that, The amino acid sequence of the aflatoxin B1 degrading enzyme mutant is shown in SEQ ID NO.

1.

2. A gene encoding a mutant of the aflatoxin B1 degrading enzyme as described in claim 1, characterized in that, The nucleotide sequence of the gene is shown in SEQ ID NO.

3.

3. A recombinant vector, characterized in that, The recombinant vector contains the gene described in claim 2.

4. A recombinant bacterium, characterized in that, The recombinant bacteria contains the gene of claim 2 or the recombinant vector of claim 3.

5. An additive, characterized in that, The additive contains the aflatoxin B1 degrading enzyme mutant as described in claim 1.

6. A feed, characterized in that, The feed contains the aflatoxin B1 degrading enzyme mutant of claim 1 or the additive of claim 5.

7. A method for preparing the aflatoxin B1 degrading enzyme mutant of claim 1, characterized in that, Includes the following steps: A recombinant expression vector containing the gene described in claim 2 was constructed, and the recombinant expression vector was introduced into a host bacterium to obtain recombinant bacteria; the recombinant bacteria were cultured in a liquid culture medium to obtain bacterial solution, and bacterial cells were obtained by centrifugation; the supernatant was collected as crude enzyme solution after ultrasonic disruption and centrifugation. The purified enzyme solution was obtained by affinity chromatography using a Ni column.

8. The application of the aflatoxin B1 degrading enzyme mutant of claim 1, the gene of claim 2, the recombinant vector of claim 3, the recombinant bacteria of claim 4, or the additive of claim 5, characterized in that, The application is for the degradation of aflatoxin B1.

9. A method for degrading aflatoxin B1, characterized in that, The aflatoxin B1 degrading enzyme mutant of claim 1 or the additive of claim 5 is added to the sample to be treated containing aflatoxin B1, and enzymatically hydrolyzed for 6-96 h at a reaction temperature of 20-90℃ and a pH of 2-9.

10. The method according to claim 9, characterized in that, The reaction conditions are enzymatic hydrolysis at 30-70℃ and pH 4-9 for 24-96 h.

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