High-temperature-resistant aflatrem-degrading enzyme, coding gene, engineering bacteria and application thereof

Abstract

The present application belongs to the field of biotechnology, and relates to a method for constructing an engineered bacterium capable of simultaneously degrading aflatoxin B1, ochratoxin A and zearalenone, in particular to a high-temperature-resistant AFB1-degrading enzyme, a coding gene, an engineered bacterium and application thereof. The engineered bacterium can produce enzymes capable of simultaneously degrading AFB1, OTA and ZEN, and the optimal temperature for degrading AFB1, OTA and ZEN is 80 DEG C and the pH is 8.5, which is a high-temperature-resistant enzyme. The degradation rate of the enzyme to AFB1 can reach 95.62%, and the degradation rates of the enzyme to OTA and ZEN are 93.80% and 86.24% respectively. The AFB1-degrading enzyme gene and the constructed engineered bacterium have great significance for the pollution of fungal toxins in food and feed products.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to a method for constructing an engineered bacterium that simultaneously degrades aflatoxin B1, ochratoxin A, and zearalenone. In particular, it refers to a heat-resistant AFB1 degrading enzyme, its encoding gene, the engineered bacterium, and its applications. Background Technology

[0002] Aflatoxin B1 (AFB1) is a secondary metabolite produced by molds such as *Aspergillus flavus* and *Aspergillus parasiticus* in moldy grains. Its structural formula contains a difuran ring and an oxanaphenone structure. The double bonds at positions 8 and 9 of the furan ring are the sites of interaction between the toxin and proteins and nucleic acids, and are key functional groups contributing to gene mutations, carcinogenesis, and teratogenesis. As early as 1993, AFB1 was classified as a Group 1 chemical carcinogen by the International Agency for Research on Cancer (IARC). Currently, aflatoxin, due to its wide contamination range and high toxicity, causes enormous losses to food security and seriously threatens human and animal health. The main methods for eliminating aflatoxin include physical, chemical, and biological methods. Among these, biological detoxification strategies are considered the most concrete, efficient, and environmentally friendly. Microbial detoxification of AFB1 includes adsorption and enzymatic degradation. In the late 1960s, the degradation of AFB1 by microorganisms was first observed in *Nocardia cordata*. Enzymatic degradation, due to its substrate specificity, high efficiency, and environmental friendliness, is widely used in the food and feed industries. Currently, many aflatoxin-degrading enzymes have been extracted and purified from various microorganisms, including bacteria, actinomycetes, and filamentous fungi. Laccase, peroxidase, reductase, and aflatoxin-degrading enzymes have been proven to degrade aflatoxin.

[0003] Ochratoxins are among the most common mycotoxins and a significant problem in food and animal feed. Within the ochratoxin family, ochratoxin A (OTA) is abundant in food and animal feed, persistent, and hazardous, posing a major threat to global public health. OTA is one of the five most important mycotoxins regulated by laws and regulations. Human exposure to OTA can lead to Balkan endemic nephropathy (BEN), chronic interstitial nephropathy (CIN), and other kidney diseases such as corticointerstitial nephropathy in humans and renal and urothelial carcinoma in rats. Ochratoxin A is presumed to cause BEN, interstitial nephritis associated with urothelial malignancies. Due to the high efficiency and safety of recombinant degrading enzymes and genetic engineering, recombinant degrading enzymes and genetic engineering are currently the preferred biological detoxification strategies for OTA.

[0004] Zearalenone (ZEN) is a fungal toxin produced by Fusarium wilt, widely distributed in corn, wheat, barley, and sorghum, causing significant yield reductions. ZEN has a dihydroxybenzoic acid ester structure, and its derivatives with different functional groups at the C1 and C6 positions of the lactone ring include α / β-zearaldehyde (α / β-ZOL), α / β-ZAL (α / β-Zal), and zearaldehyde ketone (ZAN); among them, α-ZOL and α-ZAL are more toxic than ZEN. ZEN and its derivatives accumulate in the food chain, causing serious contamination of crops and posing an increasing threat to human health, becoming a global food safety issue. As a fungal toxin, ZEN has been shown to have hepatotoxicity, hematologic toxicity, immunotoxicity, and genotoxicity. In mouse models, exposure to ZEN disrupts the reproductive hormone and testicular development system regulated by hormone-related genes.

[0005] Currently, the strategy for degrading ZEN involves using lactones or laccases to disrupt its lactone structure, supplemented by oxidation or modification of the C6 keto group and C2 or C4 hydroxyl group. Publication number CN113699126A discloses a dye decolorizing peroxidase, StDyP, for the simultaneous degradation of aflatoxin and zearalenone, achieving a degradation rate of 21.03% for aflatoxin and 45.37% for zearalenone. Publication number CN114807074A discloses an aflatoxin-degrading enzyme that can specifically degrade aflatoxin B1, achieving a degradation rate of 67.3%. Most existing enzymes specifically degrade a single toxin, while enzymes with broad-spectrum toxin degradation capabilities generally have low degradation rates. Currently, no existing enzyme can simultaneously and efficiently degrade aflatoxin B1, ochratoxin A, and zearalenone. To address the problem of mycotoxin contamination in food and feed products, our research group has conducted long-term research. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention proposes a heat-resistant AFB1 degrading enzyme, its encoding gene, an engineered bacterium, and its applications.

[0007] The technical solution of this invention is implemented as follows: The first objective of this invention is to provide a heat-resistant AFB1 degrading enzyme comprising the amino acid sequence shown in SEQ ID NO.2 or an amino acid sequence in SEQ ID NO.2 with one or more substituted, deleted or added amino acids.

[0008] Preferably, the above-mentioned heat-resistant AFB1 degrading enzyme has an amino acid sequence as shown in SEQ ID NO.2.

[0009] The second objective of this invention is to provide a gene encoding the aforementioned heat-resistant AFB1 degrading enzyme. This gene was obtained by the inventors of this application through whole-genome sequencing of Bacillus megaterium HNGD-A6, and the gene sequence of the aflatoxin degrading enzyme with a certain degree of similarity was obtained by BLASTP alignment.

[0010] Preferably, the nucleotide sequence of the above-mentioned heat-resistant AFB1 degrading enzyme gene is shown in SEQ ID NO.1.

[0011] A third objective of this invention is to provide engineered bacteria containing the aforementioned genes.

[0012] Furthermore, a method for preparing the above-mentioned engineered bacteria is provided, the steps of which are: inserting the above-mentioned gene into an intermediate vector and transforming it into Escherichia coli to obtain the engineered bacteria.

[0013] Preferably, the intermediate vector is the pET-28a(+) plasmid; and the Escherichia coli is the Escherichia coli expression strain BL21(DE3).

[0014] The fourth objective of this invention is to provide a method for preparing a thermostable AFB1 degrading enzyme using the aforementioned engineered bacteria. The steps are as follows: ligating the correctly amplified degrading enzyme gene by PCR to a plasmid, introducing it into *E. coli* DH5α, and obtaining the recombinant expression vector *E. coli* DH5α transformant after PCR and sequencing identification. After extracting the expression vector plasmid, performing bacterial PCR and double enzyme digestion verification. Introducing the verified plasmid into the *E. coli* expression strain BL21(DE3). The recombinant *E. coli* strain is then placed in a Km... r LB liquid medium enriched to OD 600 At concentrations of 0.6–0.8, IPTG induces the production of aflatoxin-degrading enzymes. The expression culture was purified using Ni-NTA to obtain the recombinant protein. The protein was concentrated using a 10 kDa ultrafiltration tube, and the concentration was determined using a total protein assay kit. The protein was then diluted with Tris-HCl to 0.4 mg / mL to obtain the thermostable AFB1 degrading enzyme.

[0015] Preferably, the concentration of kanamycin in the LB medium is 50 μg / mL, the culture conditions in the LB medium are 37℃ and 220 r / min; the final concentration of the inducer IPTG is 0.5 mmol / L, and the low-temperature induction temperature is 16℃ and the time is 16~20h.

[0016] The fifth object of this invention is to provide the application of the above-mentioned heat-resistant AFB1 degrading enzyme in the simultaneous degradation of aflatoxin B1, ochratoxin A, and zearalenone. Preferably, the heat-resistant AFB1 degrading enzyme of this application has a degradation rate of 96.52% for AFB1, a degradation rate of 93.80% for OTA, and a degradation rate of 86.24% for ZEN under the conditions of 80°C and pH 8.5.

[0017] The present invention has the following beneficial effects: 1. This application discloses a thermostable AFB1-degrading enzyme and constructs an engineered bacterium expressing this enzyme. The engineered bacterium of this invention can produce an enzyme capable of simultaneously degrading AFB1, OTA, and ZEN. The optimal temperature for the degradation of AFB1, OTA, and ZEN is 80°C and the optimal pH is 8.5, making it a thermostable enzyme. This enzyme achieves a degradation rate of 95.62% for AFB1, and degradation rates of 93.80% and 86.24% for OTA and ZEN, respectively. The AFB1-degrading enzyme gene discovered in this invention and the engineered bacterium constructed therefrom are of great significance for addressing mycotoxin contamination in food and feed products.

[0018] 2. The processing of food and feed products in industrial production is complex. The expression of the thermostable AFB1 degrading enzyme in this paper improves the enzyme's applicability in manufacturing applications. In reality, food and feed products are often contaminated by multiple mycotoxins, leading to increased toxicity. The thermostable AFB1 degrading enzyme expressed in this paper can also simultaneously degrade ochratoxin A and zearalenone. 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 This is a schematic diagram illustrating the construction principle of the recombinant expression vector in Embodiment 1 of the present invention.

[0021] Figure 2 This is an electrophoresis image after PCR amplification in Example 1 of the present invention.

[0022] Figure 3 The image shows SDS-PAGE verification images of different components in Example 2 of this invention, where M: standard protein marker; 1: uninduced; 2: whole bacterial protein; 3: supernatant protein; 4: precipitated protein.

[0023] Figure 4This is an SDS-PAGE verification image of the supernatant protein purification in Example 2 of the present invention, where M: standard protein marker; 5~10: 50, 100, 200, 300, 400, 500 mM elution buffer.

[0024] Figure 5 This is a pH optimization diagram of AttM for aflatoxin B1 in Example 3 of the present invention.

[0025] Figure 6 This is a temperature optimization diagram of AttM for aflatoxin B1 in Example 3 of the present invention.

[0026] Figure 7 This is a time-dependent degradation graph of aflatoxin B1 by AttM in Example 3 of the present invention.

[0027] Figure 8 This is a pH optimization diagram of AttM for ochratoxin A in Example 4 of the present invention.

[0028] Figure 9 This is a temperature optimization diagram of AttM for ochratoxin A in Example 4 of the present invention.

[0029] Figure 10 This is a time-dependent degradation diagram of ochratoxin A by AttM in Example 4 of the present invention.

[0030] Figure 11 This is a pH optimization diagram of AttM on zearalenone in Example 4 of the present invention.

[0031] Figure 12 This is a temperature optimization diagram of AttM on zearalenone in Example 4 of the present invention.

[0032] Figure 13 This is a time-dependent degradation graph of zearalenone by AttM in Example 4 of the present invention. 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] The following is a brief introduction to some of the biological materials, experimental reagents, and experimental equipment involved in the following examples and experimental cases: Culture medium: LB medium (L -1 ): 10g tryptone, 5g yeast extract, 10g sodium chloride, pH 7.0.

[0035] Equilibration buffer: imidazole 0.68g, disodium hydrogen phosphate 6g, Tris 1.21g, sodium chloride 16.8g, pH 8.0, bring to a final volume of 1L.

[0036] Electrode buffer: 19.4g glycine, 3g Tris, 1g SDS, bring to a final volume of 1L.

[0037] Kanamycin stock solution (100 mg / mL): Dissolve 10 g of kanamycin in 80 mL of sterile water, bring the volume up to 100 mL, filter through a 0.22 μm filter membrane, and store at -20 °C.

[0038] Elution buffer: Imidazole was added in the following amounts: 0.17025 g, 0.3405 g, 0.681 g, 1.0215 g, 1.362 g, and 1.7025 g respectively. The volume was adjusted to 50 mL with equilibration buffer, which correspond to 50 mM, 100 mM, 200 mM, 300 mM, 400 mM, and 500 mM elution buffers respectively.

[0039] Staining solution: Coomassie Brilliant Blue R250 2.5 g, methanol 450 mL, glacial acetic acid 100 mL, bring to a final volume of 1 L.

[0040] Decolorizing solution: 50 mL ethanol, 100 mL glacial acetic acid, bring to a final volume of 1 L.

[0041] In the early stages of this application, whole-genome sequencing of Bacillus megaterium HNGD-A6 was performed to obtain the whole genome sequence. The gene sequence of aflatoxin degrading enzyme with certain similarity was obtained by BLASTP alignment, as shown in SEQ ID NO.1, and named the fungal toxin degrading enzyme gene AttM.

[0042] Example 1: Construction of Recombinant Expression Vector The correctly amplified degradation enzyme gene was ligated into a plasmid and introduced into *E. coli* DH5α. After PCR and sequencing identification, the recombinant expression vector *E. coli* DH5α transformant was obtained. pET-28a(+) was selected as the vector, and *E. coli* containing the pET-28a(+) vector were inoculated onto antibiotic-free LB agar plates. After the strain grew well, single colonies were picked and placed on plates containing Km... r The plasmid was cultured overnight in resistant LB liquid medium at 37°C and 220 r / min. The plasmid was then extracted using a kit, and the plasmid concentration was determined using an ultra-micro spectrophotometer. The plasmid was stored at -20°C.

[0043] Specifically: Primers for the fungal toxin-degrading enzyme gene AttM were designed based on the genomic information of Bacillus megaterium strain HNGD-A6. Two restriction enzyme sites, SalⅠ and BamHI, were introduced at the 5' and 3' ends of the sequence, respectively. Gene primers were then designed and synthesized as follows: Upstream primer: 5'-tgcggccgcaagcttgtcgacATGATGCCAGAGATAAAGCTTTTCA-3', Downstream primer: 5'-acagcaaatgggtcgggatccTCAATAGTAACAGGCTGGCGC-3'; The target gene was amplified by PCR, and the DNA fragments of the correct size were purified using a gel extraction kit. The construction principle is as follows: Figure 1 As shown, the plasmid was double-digested with SalⅠ and BamHI, respectively. After digestion, it was ligated with the degradation enzyme gene using homologous recombinase to construct a recombinant expression vector. The recombinant product was introduced into DH5α competent cells for transformation. Plasmids were extracted from successfully transformed strains and introduced into E. coli expression strain BL21(DE3).

[0044] The PCR system, enzyme digestion system, and ligation system are shown in Tables 1, 2, and 3.

[0045] Table 1. PCR amplification system for the target gene Table 2 Double enzyme digestion system Table 3 Connection System Electrophoresis image after PCR amplification is shown below Figure 2 As shown, the size is 795 bp.

[0046] Example 2: Induced expression of degradative enzymes 1. Inducible expression of degradative enzymes (1) Select samples containing recombinant vectors E. coli BL21(DE3) single colony in 50 μg / mL Km r In LB liquid medium, it was cultured overnight at 37°C and 220 r / min.

[0047] (2) Inoculate 2% of the overnight culture into 300 mL of LB liquid medium containing Kmr, and shake and culture until its OD reaches 1. 600After reaching a concentration of 0.6–0.8, IPTG was added to a final concentration of 0.5 mmol / L, and the mixture was induced at 16°C for 16–20 h. Simultaneously, a group of bacterial cultures without added inducers was prepared under the same conditions.

[0048] (3) Aliquot the induced bacterial culture into 50 mL centrifuge tubes and centrifuge at 4000 r / min and 4℃ for 15-20 min, discarding the supernatant. Add 5 mL of equilibration buffer and resuspend the bacterial cells by pipetting to rehydrate them. Sonicate the rehydrated system under ice bath conditions, and collect the fragmented sample as whole bacterial protein for testing. Centrifuge the fragmented sample at 12000 r / min and 4℃ for 5 min, collect the supernatant as supernatant protein, and rehydrate the precipitate with equilibration buffer as precipitated protein. Samples without inducing agent are recorded as the uninduced group after sonication. Perform SDS-PAGE verification on samples of different components.

[0049] 2. Purification of degrading enzymes (1) Take out Ni-NTA and after the ethanol has flowed out, first wash the nickel column with 5 times the volume of deionized water to remove the ethanol; (2) Equilibrate the column with 5 to 10 times its volume of equilibration buffer, maintaining a flow rate of 1 mL / min; (3) After filtering the supernatant protein through a 0.45 μm filter membrane, load the sample at the same flow rate as above; (4) Elute with a prepared imidazole concentration gradient and collect the eluent; (5) The protein was detected using Coomassie Brilliant Blue staining solution. The eluent containing the protein was ultrafiltered and centrifuged to replace the buffer solution of the protein solution, thereby removing imidazole from the protein solution. The purified enzyme solution was detected by SDS-PAG gel electrophoresis.

[0050] The results of SDS-PAGE validation of samples with different components are as follows: Figure 3 As shown, whole-cell protein, supernatant protein, and precipitated protein all showed distinct bands at 30 kDa. Supernatant protein was selected for purification, and the results are shown below. Figure 4 As shown, after elution with 200 and 300 mM imidazole, a distinct and single band appeared at 30 kD.

[0051] Example 3: Degradation of AFB1 by purified enzyme The protein purified in Example 2 was concentrated using a 10 kDa ultrafiltration tube. After concentration, the protein concentration was determined using a total protein assay kit. The protein was diluted to 0.4 mg / mL with Tris-HCl and reacted with AFB1 to determine the degrading enzyme activity. The reaction system consisted of 1 mL of 1 mL containing 2.5 μg / mL AFB1, 300 mM NaCl, 50 mM NaH2PO4, 10 mM Tris base, and 20 μg protein. The reaction was carried out in the dark. After the reaction, three volumes of dichloromethane were added for extraction. The supernatant was removed, and the mixture was dried under nitrogen at 35°C. The solution was reconstituted with 0.5 mL of mobile phase, mixed, and filtered through a 0.22 μm filter before analysis. Each sample was analyzed in triplicate.

[0052] Adjust the pH of the reaction system to 5-10, then... Figure 5 It can be seen that the degradation efficiency is highest at pH 8.5, with a degradation rate of 59.43% after 24 hours.

[0053] The reaction temperature was adjusted to 30-100℃ under pH 8.5 conditions. Figure 6 It can be seen that the degradation efficiency reaches its highest level at 80℃, with a degradation rate of 86.78% after 24 hours, indicating that the enzyme in this application is a heat-resistant degradation enzyme.

[0054] Therefore, tests were conducted at pH 8.5 and 80℃ for different degradation times, and the results are as follows. Figure 7 As shown, the degradation effect gradually increases with increasing degradation time, with the highest degradation rate reaching 95.62%.

[0055] The AFB1 degradation rate is calculated using the following formula: Example 4: Degradation of OTA and ZEN by purified enzymes The protein purified in Example 2 was subjected to degradation tests with OTA and ZEN.

[0056] The reaction system of Example 3 was used, with AFB1 replaced by OTA and ZEN at the same concentrations, respectively: 1. Degradation of OTA by purified enzymes ①Reaction system OTA: 1 mL, containing 2.5 μg / mL OTA, 300 mM NaCl, 50 mM NaH2PO4, 10 mM Tris base and 20 μg protein, reacted in the dark.

[0057] ② Adjust the pH of the reaction system to 6-9 respectively, from Figure 8 As shown, the degradation efficiency is highest at pH 8.5, with a degradation rate of 73.34% after 24 hours.

[0058] ③ Adjust the reaction temperature to 30-100℃ under pH 8.5 conditions. Figure 9 It can be seen that the degradation efficiency reaches its highest level at 80℃, with a degradation rate of 81.32% after 24 hours, indicating that the enzyme in this application is a heat-resistant degradation enzyme.

[0059] ④ The degradation results of OTA were detected at 80℃ and pH 8.5 for different time periods, as shown below. Figure 10 As shown, the degradation rate of OTA within 24 hours is 81.32%, reaching a maximum of 93.8% at 72 hours.

[0060] 2. Degradation of ZEN by purified enzymes ① Reaction system: 1 mL ZEN, containing 2.5 μg / mL ZEN, 300 mM NaCl, 50 mM NaH2PO4, 10 mM Tris base, and 20 μg protein. The reaction was carried out in the dark. After the reaction, 3 times the volume of dichloromethane was added for extraction. After removing the supernatant, the mixture was dried under nitrogen at 35°C. The solution was reconstituted with 0.5 mL of mobile phase, mixed well, and filtered through a 0.22 μm filter before analysis. Each sample was analyzed three times.

[0061] ② Adjust the pH of the reaction system to 6-9 respectively, from Figure 11 As shown, the degradation efficiency is highest at pH 8.5, with a degradation rate of 60.3% after 24 hours.

[0062] ③ Adjust the reaction temperature to 30-100℃ under pH 8.5 conditions. Figure 12 It can be seen that the degradation efficiency reaches its highest level at 80℃, and the degradation rate is 65% after 24 hours, indicating that the enzyme in this application is a heat-resistant degradation enzyme.

[0063] ④ The degradation results of OTA were detected at 80℃ and pH 8.5 for different time periods, as shown below. Figure 13 As shown, the ZEN degradation rate was 67.82% within 24 hours, reaching a maximum of 86.24% at 72 hours.

[0064] 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. The application of a heat-resistant AFB1 degrading enzyme in the simultaneous degradation of aflatoxin B1, ochratoxin A, and zearalenone, characterized in that: The amino acid sequence of the heat-resistant AFB1 degrading enzyme is shown in SEQ ID NO.2; At 80℃ and pH 8.5, the degradation rate of AFB1 was 96.52%, the degradation rate of OTA was 93.80%, and the degradation rate of ZEN was 86.24%.

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