A zearalenone-degrading enzyme, a gene encoding the same and use thereof

Abstract

The application discloses a zearalenone degrading enzyme and a coding gene and application thereof, and belongs to the field of enzyme engineering. The amino acid sequence of the zearalenone degrading enzyme is shown as SEQ ID NO:2, and the nucleotide sequence of the coding gene is shown as SEQ ID NO:1. The zearalenone degrading enzyme PRS provided by the application is a brand-new enzyme, has good temperature and pH stability, has strong degradation capacity for the mycotoxin zearalenone, has a fast degradation speed, and can be widely applied to zearalenone enzymolysis. It can be seen that the application provides a new enzyme preparation for the biological degradation of zearalenone, the enzyme can be used in the industrial fields of agriculture, feed and food, and has good application potential, thereby laying a good technical foundation for ensuring feed safety and reducing the harm caused by zearalenone pollution to human and animal health.

Description

Technical Field

[0001] This invention relates to the field of enzyme engineering, and in particular to a zearalenone-degrading enzyme, its encoding gene, and its applications. Background Technology

[0002] Zearalenone (ZEN) is an estrogenic fungal toxin with strong heat resistance, requiring 110℃ for 1 hour to be completely destroyed. It primarily contaminates crops such as wheat, barley, oats, and corn, as well as animal feed. Chen Daofu et al. selected 86 feed samples from Henan Province and found that the exceedance rate of zearalenone was 43.59%, with the highest content reaching 11900 μg / kg. Zearalenone mainly affects the reproductive system, causing premature maturity, abnormal reproductive function, and even death in breeding pigs or poultry. Therefore, how to effectively control and eliminate zearalenone contamination in food and feed has become an urgent problem to be solved in the food and feed industries.

[0003] Currently, the detoxification technology of zearalenone has attracted much attention. It mainly utilizes physical, chemical, and biological detoxification methods. Among these, biological detoxification utilizes certain functional microorganisms to adsorb toxins, or their metabolites, such as secreted enzymes, to decompose toxins, thereby reducing the ZEN content or toxicity in products. This method is low-cost, highly efficient, highly specific, pollution-free, and does not damage the raw material components, offering advantages that are difficult to replace with other methods, and has gradually become a research hotspot. Previous studies have reported extensively on the ZEN degradation role of fungi. Aspergillus, Penicillium, Rhizopus, Trichosporon, and other fungi and their metabolites have been found to have the ability to degrade ZEN and catalyze the formation of α-zearalenol, β-zearalenol, and Zearalenone-4-O-Sulfate. Therefore, utilizing biotechnology to degrade and detoxify ZEN and its derivatives is a major approach to solving this problem in the future.

[0004] Biotechnological detoxification mainly utilizes two pathways: bacterial degradation and enzymatic degradation. Among these, the enzymes capable of degrading zearalenone (ZEN) fall into three main categories: laccases, peroxidases, and lactone hydrolases. To date, several enzymes capable of specifically binding to and degrading ZEN have been identified. However, research on enzymes related to ZEN degradation in screened microorganisms remains limited, and the number of discovered ZEN-degrading enzymes is relatively small compared to other mycotoxins. Furthermore, most ZEN-degrading enzymes suffer from low activity and poor pH stability, restricting their application. To expand our understanding of ZEN detoxification enzymes and promote the development of protein engineering, further exploration of novel ZEN-degrading enzymes is essential. Summary of the Invention

[0005] The purpose of this invention is to provide a zearalenone-degrading enzyme, its encoding gene, and its applications to solve the problems existing in the prior art. The zearalenone-degrading enzyme PRS provided by this invention is a novel enzyme with good temperature and pH stability, strong degradation ability against the fungal toxin zearalenone, and fast degradation speed. It can be widely used in the enzymatic hydrolysis of zearalenone.

[0006] To achieve the above objectives, the present invention provides the following solution:

[0007] The present invention provides a zearalenone-degrading enzyme, the amino acid sequence of which is shown in SEQ ID NO: 2.

[0008] The present invention also provides a gene encoding a zearalenone-degrading enzyme in zearalenone, the nucleotide sequence of which is shown in SEQ ID NO: 1.

[0009] The present invention also provides a recombinant expression vector containing the above-mentioned coding gene.

[0010] The present invention also provides a recombinant bacterium comprising the above-described recombinant expression vector.

[0011] The present invention also provides the application of the above-mentioned zearalenone-degrading enzyme, the above-mentioned encoding gene, the above-mentioned recombinant expression vector, or the above-mentioned recombinant bacteria in the degradation of zearalenone.

[0012] The present invention also provides the application of the above-mentioned zearalenone-degrading enzyme, the above-mentioned encoding gene, the above-mentioned recombinant expression vector, or the above-mentioned recombinant bacteria in the preparation of a zearalenone-degrading agent.

[0013] Furthermore, a recombinant expression vector containing the coding gene is constructed, the recombinant expression vector is transformed into a host bacterium to obtain a recombinant bacterium, the recombinant bacterium is induced and cultured, and the zearalenone-degrading enzyme is harvested to degrade zearalenone.

[0014] The present invention also provides a zearalenone degradation formulation comprising the above-mentioned zearalenone degrading enzyme.

[0015] The present invention also provides a method for degrading zearalenone, comprising the step of mixing and reacting the above-mentioned zearalenone-degrading enzyme or the above-mentioned degradation agent with zearalenone.

[0016] The present invention discloses the following technical effects:

[0017] This invention utilizes a culture medium with zearalenone as the sole carbon source to screen zearalenone-degrading bacteria. Using the genome of the screened zearalenone-degrading bacterium (Alcaligenesfaecalis) as a template, primers were designed for PCR amplification, and the protein gene with the ability to degrade zearalenone was obtained.

[0018] The zearalenone-degrading enzyme PRS obtained in this invention is a novel enzyme with good temperature and pH stability, strong degradation ability against the mycotoxin zearalenone, and rapid degradation rate. It can be widely used in the enzymatic hydrolysis of zearalenone. Therefore, this invention provides a new enzyme preparation for the biodegradation of zearalenone. This enzyme has good application potential in agricultural, feed, and food industries, thus laying a good technical foundation for ensuring feed safety and reducing the harm to human and animal health caused by zearalenone contamination. 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 embodiments 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 graph showing the SDS-PAGE electrophoresis analysis results of the PRS gene induced in E. coli in Example 3;

[0021] Figure 2 The image shows the HPLC detection of PRS degradation of zearalenone in Example 3; the top image shows zearalenone (standard) (concentration 10 μg / mL); the bottom image shows zearalenone degrading enzyme PRS + zearalenone, reaction for 30 min.

[0022] Figure 3 This is a bar chart showing the degradation of zearalenone by PRS in Example 3. Detailed Implementation

[0023] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0024] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0025] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0026] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0027] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0028] Example 1

[0029] This embodiment specifically illustrates the screening method for zearalenone-degrading bacteria.

[0030] (1) Enrichment of strains

[0031] Weigh 5g of soil samples and add them to 250mL Erlenmeyer flasks containing 100mL of sterile water. Resuspend and shake for 1 hour. Then, with an inoculum volume of 1mL, transfer the sample suspension to 100mL of zearalenone enrichment medium containing 10mg / mL ZEN toxin. Incubate at 30℃ and 180rpm for 7 days. Subsequently, transfer the bacterial suspension to 100mL of MSM medium containing 20μg / mL ZEN toxin with an inoculum volume of 1mL. Repeat this transfer process 4 times, continuously increasing the ZEN concentration, until the final ZEN concentrations in the medium are 10μg / mL, 20μg / mL, 30μg / mL, and 40μg / mL, respectively.

[0032] (2) Plate method for screening zearalenone-degrading bacteria

[0033] Enrichment and domestication of the degrading bacterial flora: 200 μL of bacterial suspension was spread onto inorganic salt culture plates using zearalenone as the sole carbon source. Observations were made daily, and vigorous fungi were streaked three times on PDA agar plates. The PDA plates were incubated upside down at 25°C, and single colonies were collected for purification. The selected strain could grow using zearalenone as the sole carbon source, indicating its potential for zearalenone degradation. Sequencing and identification of the strain, using 16S rDNA, identified it as *Alcaligenes faecalis*.

[0034] The enrichment medium containing zearalenone consisted of 1.52 g / L KH2PO4, 2.44 g / L Na2HPO4, 0.2 g / L MgSO4·7H2O, 0.5 g / L (NH4)2SO4, 0.06 g / L CaCl2, 10.0 g / L NaCl, and 10 μg / mL zearalenone.

[0035] (3)Save

[0036] The isolated strains were purified and attached to sterilized filter paper. After the strains had grown on all the filter paper, the filter paper containing the target strains was collected, air-dried, and then transferred to -20℃ for acclimatization, and then transferred to -80℃ for storage.

[0037] Example 2: Cloning method of the zearalenone-degrading enzyme PRS gene

[0038] Using the total DNA of the *Alcaligenes faecalis* strain obtained in Example 1 as a template, primers were designed: the upstream primer was PRSf: 5'-cgggatccATGGCTACACTTCGTCTGG-3', and the downstream primer was PRSr: 5-tcccccgggCTTGTTAGGCTGAGGCGTC-3'. The PCR amplification reaction system was 10 μL: 1 μL of total DNA template, 0.5 μL each of upstream and downstream primers PRSf and PRSr, 5 μL of Taq DNA polymerase, and 3 μL of ddH2O. The PCR amplification program was: 95℃ denaturation for 45 s, 55℃ annealing for 45 s, 72℃ extension for 1 min, for a total of 30 cycles; 72℃ extension for 5 min, and storage at 4℃. After the amplification reaction, the PCR products were detected: 5 μL of PCR product was added to the sample buffer and loaded onto the wells of a 1% agarose gel. After electrophoresis, the results were checked and photographed using a gel imaging system. The target DNA fragment was excised and recovered from the gel using a PCR purification kit. Sequencing of the PCR product revealed the nucleotide sequence of the PRS gene as shown in SEQ ID NO: 1:

[0039] ATGGCTACACTTCGTCTGGGTGACCTGGCCCCTGATTTTGAACAAAAATCCTCCGCTGGCACCATCAAGTTTCACGAGTTCTTGGGTAATAGCTGGGGCGTCTTGTTCTCCCACCCAGCCGACTTTACGCCTGTGTGCACCACCGAACTGGGTTACACCGCCAAGCTGGCGGACGAGTTCGCCAAGCGCGGCGTGAAAGTGATTGCCGTTTCGGTTGACGATGTTGATTCCCACGGCAAGTGGATTGATGACATCAACGAAACTCAAAAAACCACCGTTAACTTCCCCATCCTGGCTGATGCGGACCGCAAGGTGTCGGATCTGTACGATATGATCCACCCCAACGCCAGCACCACCGTGACTGTGCGCTCGGTGTTCATCATCGACCCGGCCAAGAAAGTGCGTCTGATCATCACCTACCCAGCCAGCACGGGCCGCAACTTCAACGAAATCCTGCGCGTGATCGATTCCCTGCAACTGACCGACAGCCACAGCGTGGCCACGCCAGTGAACTGGGAAGACGGTGATGACGTGATCATTGTTCCTAGCCTGAAAGATCCTGAAGTTCTGAAGCAGAAATTCCCCAAAGGCTACACCGAGATCCGTCCTTACCTGCGTGTGACGCCTCAGCCTAACAAG (SEQ ID NO: 1).

[0040] The amino acid sequence of the protein encoded by the PRS gene is shown in SEQ ID NO: 2:

[0041] MATLRLGDLAPDFEQKSSAGTIKFHEFLGNSWGVLFSHPADFTPVCTTELGYTAKLADEFAKRGVKVIAVSVDDVDSHGKWIDDINETQKTTVNFPILADADRKVSDLYDMIHPNASTTVTVRSVFIIDPAKKVRLIITYPASTGRNFNEILRVIDSLQLTDSHSVATPVNWEDGDDVIIVPSLKDPEVLKQKFPKGYTEIRPYLRVTPQPNK (SEQ ID NO: 2).

[0042] Example 3

[0043] Expression and functional identification of the zearalenone-degrading enzyme PRS gene in Escherichia coli

[0044] (1) Constructing expression vectors

[0045] Primers were designed based on the zearalenone-degrading enzyme PRS encoding gene obtained in Example 2. The primers contain restriction enzyme sites (BamhI, NotI), and the primers are as follows:

[0046] Primer 1: 5'-CGGGATCCATGAAAACTGTTGGCGACAAACT-3';

[0047] Primer 2: 5'-ATAAGAATGCGGCCGCCAGCGTGGCGCCGCCAACC-3'.

[0048] The PCR reaction system (50 μL) consisted of: 25 μL of 2×primestar-max, 1 μL of primer 1, 1 μL of primer 2, 1 μL of template DNA, and double-distilled water to a final volume of 50 μL.

[0049] The PCR amplification conditions were: pre-denaturation at 95℃ for 3 min, followed by 30 cycles (denaturation at 95℃ for 15 s, annealing at 60℃ for 30 s, extension at 72℃ for 5 min), and cooling to 4℃.

[0050] After PCR amplification of the PRS gene fragment, the PCR product and the pET-28a(+) vector were double-digested with BamhI and NotI, respectively. The digested PCR product was purified and ligated into the pET-28a(+) vector using T4 DNA ligase. This ligation was then performed on competent E. coli BL21(DE3) cells, plated on solid LB medium (containing 50 μg / mL kanamycin sulfate), and single colonies were picked and inoculated into LB liquid medium (containing 50 μg / mL kanamycin sulfate). The transformation results were detected using double digestion and sequencing. E. coli successfully transformed with the pET-28a(+)-PRS plasmid were considered positive transformants, namely E. coli BL21(DE3) / pET-28a(+)-PRS.

[0051] (2) Induced expression

[0052] The cells transformed with E. coLiBL21(DE3) / pET-28a(+)-PRS were cultured in LB medium (containing 50 μg / mL kanamycin sulfate) (37℃, 200 rpm). OD 600Once the pH reached 0.6, 1.0 mmol / L IPTG was added, and the culture was continued at 37°C for 4 hours. The liquid culture was then centrifuged for 10 minutes (12000 rpm, 4°C), and the supernatant was discarded to obtain the recombinant *E. coli* precipitate. The cells were resuspended in 30 mL of cell lysis buffer, and the cells were disrupted using an ultrasonic cell disruptor (frequency 300 Hz, sonication for two seconds, pause for two seconds, for a total of 15 minutes, followed by an ice bath). The cells were centrifuged for 10 minutes (12000 rpm, 4°C), cell debris was removed, and the supernatant was collected; this was the crude PRS enzyme solution for recombinant expression.

[0053] The crude enzyme solution was placed on ice. Based on the presence of a His-tag in the expressed recombinant enzyme, affinity chromatography was performed using a Ni column. After equilibrating the Ni column at a flow rate of 1 mL / min, the resuspended crude enzyme solution was directly loaded onto the sample at a flow rate of 0.5 mL / min. Unadsorbed or adsorbed nonspecific proteins were eluted with buffer (pH 7.4, 50 mM NaCl, containing 10 mM imidazole) at a flow rate of 1 mL / min. The target protein was collected by elution with buffer (pH 7.4, 50 mM NaCl, 300 mM imidazole) to obtain the purified enzyme solution.

[0054] (3) SDS-PAGE electrophoresis analysis of recombinant PRS crude enzyme solution and purified enzyme solution.

[0055] Take 1 mL of the crude recombinant PRS enzyme solution obtained in step (2) and 1 mL of the purified enzyme solution, analyze them by 12.5% ​​(w / v) SDS-PAGE electrophoresis, and stain with Coomassie brilliant blue.

[0056] SDS-PAGE protein electrophoresis buffer: 10% (w / v) sodium dodecylbenzenesulfonate (SDS) solution; 1% (v / v) tetramethylethylenediamine (TEMED); 10% (w / v) ammonium persulfate (AP);

[0057] Sample dissolution solution: 2% (w / v) SDS, 5% (v / v) mercaptoethanol, 10% (v / v) glycerol, 0.02% (w / v) bromophenol blue, 0.01 mol / L pH 8.5 Tris-HCl buffer;

[0058] Gel stock solution: 30g acrylamide (Acr) + 0.8g methylenebisacrylamide (Bis), dilute to 100mL with deionized water, filter, protect from light, and store at 4℃ for one month.

[0059] Separating gel formulation: 6.22 mL monomer (30% (w / v) acrylamide, 0.8% (w / v) crosslinking agent), 5.0 mL buffer (3 M Tris, 0.3% (w / v) SDS, pH 8.45), 2.38 mL distilled water, 2.0 mL glycerol, 50 μL 10% (w / v) AP, 10 μL LTEMED;

[0060] Stacking gel formulation: 1.62 mL monomer, 3.10 mL buffer, 7.78 mL distilled water, 45 μL 10% (w / v) AP, 15 μL LTEMED;

[0061] Gel buffers: separating gel buffer and stacking gel buffer (3.0 mol / L 0.3% (w / v) pH 8.45 Tris-HCl buffer);

[0062] Electrode buffer (pH 8.3): Tris 30.3g + glycine 188g + SDS 10g, bring the volume to 1000mL, and dilute 10 times before use.

[0063] Staining solution: Coomassie Brilliant Blue R250 0.29g.

[0064] Electrophoresis results as follows Figure 1 As shown, the zearalenone degrading enzyme PRS in zearalenone can be induced to express in large quantities in E. coli BL21(DE3) / pET-28a(+)-PRS strain, and a clear protein band (at 27.1 kDa) appears.

[0065] (4) Tests for degradation of zearalenone by the expressed product

[0066] The PRS purified enzyme solution obtained in step (2) was used to test the degradation of zearalenone. For the isolated and purified enzyme, its activity was directly determined by its use in the catalytic degradation reaction of ZEN. The enzyme used for activity determination was diluted to a 0.1 mg / mL enzyme solution for later use. The reaction system contained 2 μL of 5 mg / mL ZEN acetonitrile solution, 50 μL of enzyme solution (0.1 mg / mL), and 448 μL of Tris-HCl solution. The reaction was carried out at 30 °C for 24 h, and then 500 μL of methanol was added to terminate the reaction. The ZEN concentration of the solution after the reaction was determined by HPLC.

[0067] The HPLC analysis conditions were as follows: the detector was a Dionex UV detector, the column was a C18 column (250 mm × 4.6 mm, 5 μm), the UV absorption wavelength was 264 nm, the column temperature was 35 ℃, the mobile phase was 60% vol acetonitrile aqueous solution (ultrapure water), the flow rate was 1.0 mL / min, and the injection volume was 10 μL. A standard curve of zearalenone content versus peak area was also plotted. Zearalenone standards with different concentration gradients were prepared, filtered through a 0.45 μm pore size filter, and then loaded onto the sample for detection of residual zearalenone. The results showed that the prokaryotic recombinant expression PRS pure enzyme solution could degrade 41% of zearalenone in about 30 min. Figure 2 and Figure 3As shown (Note: The standard curve formula for zearalenone-peak area is y = 82.234x + 28.165. Substituting the peak area into the y value, we can find x as the zearalenone content in μg / mL. According to...) Figure 3 Peak area values ​​yielded a gibberellic acid (GAA) content of 6.175 μg / mL in the control group and 3.694 μg / mL in the experimental group. This resulted in a degradation rate of approximately 41%.

[0068] Different metal ions were added to zearalenone enrichment medium containing 10 mg / mL ZEN toxin, and then 300 μL of the PRS purified enzyme solution obtained in step (2) was added to the zearalenone enrichment medium. A zearalenone enrichment medium containing the same concentration of ZEN was used as a control group. Three replicates were set for each sample, as shown in Table 1. The reaction was carried out at 30 °C for 24 h, and then 500 μL of methanol was added to terminate the reaction. The ZEN concentration in the resulting solution was determined by HPLC.

[0069] Table 1 Effects of metal ions on enzyme activity

[0070]

[0071] Table 1 shows the results for Te. 3+ and Zn 2+ It has no effect on the enzyme activity of PRS, Mn 2+ It has little effect on the enzyme activity of PRS, Mg 2 + NH 4+ and Cu 2+ It has a relatively significant inhibitory effect on the enzyme activity of PRS.

[0072] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. The application of a zearalenone-degrading enzyme or a gene encoding a zearalenone-degrading enzyme, or a recombinant expression vector containing said gene, or a recombinant bacterium, in the degradation of zearalenone, characterized in that, The amino acid sequence of the zearalenone degrading enzyme is shown in SEQ ID NO: 2; the nucleotide sequence of the encoding gene is shown in SEQ ID NO:

1.

2. The application of a zearalenone-degrading enzyme or a gene encoding a zearalenone-degrading enzyme, or a recombinant expression vector or recombinant bacteria containing said gene, in the preparation of a zearalenone-degrading agent, characterized in that, The amino acid sequence of the zearalenone degrading enzyme is shown in SEQ ID NO: 2; the nucleotide sequence of the encoding gene is shown in SEQ ID NO:

1.

3. The application according to claim 2, characterized in that, A recombinant expression vector containing the encoded gene was constructed, and the recombinant expression vector was transformed into a host bacterium to obtain recombinant bacteria. The recombinant bacteria were induced and cultured to harvest the zearalenone-degrading enzyme for degrading zearalenone.

4. A method for degrading zearalenone, characterized in that, The method includes the step of mixing and reacting zearalenone-degrading enzyme and zearalenone; the amino acid sequence of the zearalenone-degrading enzyme is shown in SEQ ID NO: 2.

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