Zearalenone degrading enzyme as well as coding gene and application thereof
By developing and expressing a novel zearalenone-degrading enzyme and its encoding gene, the problem of insufficient activity of existing enzymes has been solved, achieving efficient and stable degradation of zearalenone, which is suitable for agriculture, feed and food industries, and ensures safety.
Patent Information
- Application Number
- CN202511470112.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-10-15
- Publication Date
- 2026-02-27
AI Technical Summary
Existing zearalenone-degrading enzymes cannot meet industrial needs, enzyme activity needs to be improved, and biodegradation methods have limited efficiency in treating zearalenone.
A novel zearalenone-degrading enzyme and its encoding gene were developed. By expressing and purifying the enzyme in Escherichia coli, a zearalenone-degrading enzyme with high enzyme activity and stability was obtained for the preparation of degradation formulations and application in the biodegradation of zearalenone.
It achieves efficient degradation of zearalenone, with a fast degradation rate and good temperature and pH stability, making it suitable for agriculture, feed and food industries, ensuring feed safety and reducing health hazards.
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Abstract
Description
Technical Field
[0001] This invention relates to a zearalenone-degrading enzyme in zearalenone, its encoding gene, and its applications, belonging to the field of biotechnology. Background Technology
[0002] Zearalenone (ZEN) was first isolated from maize infected with Fusarium by Stob M et al. Formerly known as F-2 toxin, it is a non-steroidal estrogenic fungal toxin biosynthesized by various Fusarium fungi via the polyketide pathway, including *Fusarium graminearum*, *Fusarium stoloniferum*, *Fusarium cremastrae*, and *Fusarium equisetifolium*, which are common soil fungi in temperate and warm-climate countries.
[0003] ZEN possesses a phenolic dihydroxy lactone structure similar to that of endogenous animal estrogens, allowing it to competitively bind to estrogen receptors. Due to this structural similarity, ZEN may trigger a series of estrogen-like biological effects in vivo, potentially impacting normal physiological functions. Consuming food contaminated with ZEN and its derivatives, which is absorbed and metabolized by the liver and intestines, can lead to reproductive, immune, and genotoxic effects, as well as carcinogenicity.
[0004] Currently, various methods exist for the degradation of ZEN and its derivatives, including physical, chemical, and biological detoxification. Physical methods include heat treatment, radiation, and adsorption; chemical methods include alkaline hydrolysis, ozone treatment, hydrogen peroxide treatment, and vitamin E treatment. However, both of these degradation methods have limitations, such as easily destroying the nutrients in feed or grains, and the potential for chemical residues causing secondary pollution. Compared to these two methods, biological degradation is green, efficient, and highly targeted in its detoxification process, showing great promise for future development.
[0005] In biodegradation methods, one approach is to remove toxins through direct adsorption by microbial cells; another is to utilize secondary metabolites or specific enzymes produced by microorganisms during their growth and metabolism to degrade ZEN, thereby generating degradation products with lower or no toxicity. Biological detoxification not only offers feasibility for large-scale operation but also ensures that feed nutrients are not compromised. Its high specificity and efficiency give it significant advantages in ZEN treatment, while also being environmentally friendly, effectively preventing secondary pollution. However, the zearalenone-degrading enzymes currently used in biodegradation methods cannot meet the demands of industrial-scale production. There is an urgent need to develop new degrading enzymes and to obtain highly efficient and stable zearalenone-degrading enzymes through modification or screening to overcome the shortcomings of existing technologies. Summary of the Invention
[0006] To address the shortcomings of the existing technology, this invention provides a zearalenone-degrading enzyme, its encoding gene, and its applications, aiming to solve the technical problem that the enzyme activity of the zearalenone-degrading enzyme used in current biodegradation methods needs to be further improved.
[0007] The first technical solution provided by the present invention is a zearalenone degrading enzyme, the amino acid sequence of which is shown in SEQ ID NO:2.
[0008] The second technical solution provided by the present invention is a gene encoding the zearalenone-degrading enzyme described in the first technical solution.
[0009] In some embodiments, the nucleotide sequence of the gene is shown in SEQ ID NO:1.
[0010] The third technical solution provided by the present invention is a recombinant plasmid carrying the gene described in the second technical solution.
[0011] In some embodiments, the recombinant plasmid is expressed using plasmid pET-28a as an expression vector.
[0012] The fourth technical solution provided by the present invention is to express the zearalenone-degrading enzyme of the first technical solution, or to contain the gene of the second technical solution, or to transform recombinant cells with the recombinant plasmid of the third technical solution.
[0013] In some embodiments, the recombinant cells use Escherichia coli as a host.
[0014] In some embodiments, the Escherichia coli is Escherichia coli BL21(D3).
[0015] The fifth technical solution provided by the present invention is a degradation agent for zearalenone, wherein the degradation agent contains the zearalenone degradation enzyme described in the first technical solution.
[0016] The sixth technical solution provided by this invention is a method for degrading zearalenone, wherein the method involves adding the zearalenone-degrading enzyme described in the first technical solution or the degradation agent described in the fifth technical solution to a system containing zearalenone.
[0017] The present invention provides a seventh technical solution, which is the application of the zearalenone-degrading enzyme described in the first technical solution, the gene described in the second technical solution, the recombinant plasmid described in the third technical solution, or the recombinant cell described in the fourth technical solution in the preparation of a product containing zearalenone-degrading enzyme.
[0018] The present invention provides an eighth technical solution, which is the application of the zearalenone-degrading enzyme described in the first technical solution, the gene described in the second technical solution, the recombinant plasmid described in the third technical solution, the recombinant cell described in the fourth technical solution, or the degradation agent described in the fifth technical solution in the degradation of zearalenone.
[0019] Compared with the prior art, the beneficial effects of the present invention are as follows: The zearalenone-degrading enzyme 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
[0020] Figure 1 SDS-PAGE electrophoresis images of zearalenone degrading enzyme Z12 protein before and after purification; M: protein marker; Lane 1: crude Z12 protein supernatant; Lane 2: purified Z12 protein; Lane 3: Z12 protein precipitate.
[0021] Figure 2 The optimal pH curve for zearalenone-degrading enzyme Z12 in zearalenone is shown.
[0022] Figure 3 The optimal temperature curve for zearalenone-degrading enzyme Z12 in zearalenone is shown.
[0023] Figure 4 HPLC chromatogram of zearalenone degradation by zearalenone-degrading enzyme Z12. Detailed Implementation
[0024] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explanation of the present invention and are not intended to limit the present invention.
[0025] Test method: Zearalenone-degrading enzyme activity assay: The amount of enzyme required to reduce 1 μg of substrate per minute is recorded as 1 enzyme activity unit (U). The total reaction volume is 250 μL, containing 5 μL of enzyme solution (0.5 mg / mL), 5 μL of ZEN standard solution (5 mg / mL), and 240 μL of Tris-HCl buffer. The reaction is carried out at the optimum temperature for 10 min. The reaction is stopped by adding 750 μL of methanol.
[0026] Determination of zearalenone in zearalenone: The content of ZEN was determined by high performance liquid chromatography (HPLC). A 20 μL sample was analyzed by HPLC (Agilent 1260). Chromatographic conditions included: UV detector at 274 nm; mobile phase of 60% acetonitrile (1 mL / min); and column temperature of 30 °C.
[0027] Raw materials used in the examples: Recombinant strains were cultured and expressed on LB medium. Restriction endonucleases and DNA polymerases were purchased from TaKaRa; mini plasmid extraction kits, homologous recombination kits, and gel extraction kits were purchased from Nanjing Novizan Biotechnology Co., Ltd.
[0028] Example 1 1. Artificial synthesis of gene sequences The nucleotide sequence encoding the zearalenone degrading enzyme gene (as shown in SEQ ID NO:1) was artificially synthesized by Suzhou Jinkairui Biotechnology Co., Ltd. using conventional techniques in the field. The gene was inserted into the plasmid vector pUC-GW and stored for later use.
[0029] 2. Gene sequence amplification Based on the nucleotide sequence shown in SEQ ID NO: 1, the following primer pair was designed: Forward primer: 5′-ATGGATAGCACAAGAACAAGAAGTACAGTTA -3′ Reverse primer: 5′-TTACTTGTTCTCGAGATATTTGCGCG -3′.
[0030] Amplification was performed using the PCR system and primer pairs listed in Table 1 to obtain the amplification products.
[0031] Table 1 PCR system
[0032] 3. Construction of recombinant expression vectors A 10 μL homologous recombination system was constructed, and the reaction components are shown in Table 2. After reacting the above system in a 50℃ water bath for 15–20 min, the recombinant plasmid pET-28a- was obtained. z12 The recombinant plasmid pET-28a- z12 join in E. coli BL21 competent cells were chemically transformed and cultured overnight in an inverted incubator at 37°C.
[0033] Table 2 Homologous recombination system
[0034] 4. Preparation of zearalenone-degrading enzyme The obtained single colonies were verified by colony PCR to obtain correctly verified bacteria and recombinant cells. E. coli BL21-pET-28a- z12 Recombinant cells E. coli BL21-pET-28a- z12 Single colonies were inoculated into 10 mL of LB liquid medium (containing 100 mg / L kanamycin) and cultured overnight at 37°C and 220 r / min. The next day, the colonies were inoculated into 50 mL of liquid medium (containing 100 mg / L kanamycin) and cultured at 37°C and 220 r / min until OD600 = 0.6 to 0.8. IPTG was added to a final concentration of 0.8 mmol / L and cultured at 20°C and 220 r / min for 20 h. The cells were then collected at 4°C and 6000 r / min. An equal volume of Tris-HCl buffer (pH = 7.0) was added, and the cells were sonicated to disrupt the cell walls. The supernatant was collected at 4°C and 12000 r / min as the crude enzyme solution, which was then purified by Ni-NTA to obtain the purified enzyme solution.
[0035] The zearalenone-degrading enzyme gene can be expressed solublely in *Escherichia coli* BL21(DE3). For example... Figure 1 As shown, the polyacrylamide gel electrophoresis (SDS-PAGE) results showed a single protein band in the 25kDa-33kDa range, indicating that the degrading enzyme was successfully expressed in E. coli BL21(DE3). Lane 2 in Figure 1 shows the purified recombinant protein, which contains the purified degrading enzyme.
[0036] Example 2: Determination of the enzymatic properties of zearalenone-degrading enzymes 1. Optimal pH of zearalenone-degrading enzyme The crude enzyme solution obtained in Example 1 was purified using a nickel column to obtain pure enzyme. The enzyme was then reacted at the same temperature in phosphate buffer (pH 5.0–7.0), Tris-HCl buffer (pH 7.0–9.0), and glycine-NaOH buffer (pH 9.0–12.0), and the relative enzyme activity at different pH values was measured. The results are as follows: Figure 2 As shown, the enzyme activity at the optimal pH is considered to be 100%. It can be seen that the optimal pH range for zearalenone-degrading enzyme is 7.0–8.0.
[0037] 2. Optimal temperature for zearalenone-degrading enzymes The crude enzyme solution obtained in Example 1 was purified using a nickel column to obtain pure enzyme. The relative enzyme activity at different temperatures was measured at selected temperature points (intervals of 5°C or 10°C) within the range of 20°C to 70°C. The results are as follows: Figure 3As shown, the enzyme activity at the optimal temperature is considered to be 100%. It can be seen that the optimal temperature range for zearalenone-degrading enzyme is 37℃.
[0038] Example 3: Degradation by zearalenone-degrading enzymes The reaction was carried out at 37°C in a degradation system with a pH of 8. The specific degradation system was as follows: the total reaction volume was 250 μL, containing 5 μL of enzyme solution (0.5 mg / mL), 5 μL of ZEN standard solution (5 mg / mL) and 240 μL of Tris-HCl buffer. Under optimal conditions, the enzyme activity of Z12 degrading enzyme reached 176.53 U / mg, and the degradation rate of ZEN was 84.25% within half an hour.
[0039] The HPLC results of ZEN standard sample and ZEN degradation by Z12 degrading enzyme are as follows: Figure 4 As shown.
[0040] While existing zearalenone-degrading enzymes such as ZenH have some degradation activity, their degradation rate is still limited to 75.7% (Hu J, Wang G, Hou M, et al. New Hydrolase from Aeromicrobium sp. HA for the Biodegradation of Zearalenone: Identification, Mechanism, and Application[J]. Journal of Agricultural and Food Chemistry, 2023.DOI:10.1021 / acs.jafc.2c06410.). The Z12 degrading enzyme provided by this invention can achieve a higher zearalenone degradation rate under the same conditions, thereby significantly improving the detoxification efficiency.
[0041] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.
Claims
1. A zearalenone-degrading enzyme, characterized in that, The amino acid sequence of the zearalenone degrading enzyme is shown in SEQ ID NO:
2.
2. The gene encoding the zearalenone-degrading enzyme of claim 1.
3. A recombinant plasmid carrying the gene described in claim 2.
4. The recombinant plasmid according to claim 3, characterized in that, The recombinant plasmid uses plasmid pET-28a as the expression vector.
5. Recombinant cells expressing the zearalenone-degrading enzyme of claim 1, or containing the gene of claim 2, or transformed with the recombinant plasmid of claim 3 or 4.
6. The recombinant cell according to claim 5, characterized in that, The recombinant cells used Escherichia coli as the host.
7. A degradation agent for zearalenone, characterized in that, The degradation agent contains the zearalenone-degrading enzyme as described in claim 1.
8. A method for degrading zearalenone, characterized in that, The method involves adding the zearalenone-degrading enzyme of claim 1 or the degradation agent of claim 7 to a system containing zearalenone.
9. The use of the zearalenone-degrading enzyme of claim 1, the gene of claim 2, the recombinant plasmid of claim 3 or 4, or the recombinant cell of claim 5 or 6 in the preparation of a product containing zearalenone-degrading enzyme.
10. The use of the zearalenone-degrading enzyme of claim 1, the gene of claim 2, the recombinant plasmid of claim 3 or 4, the recombinant cell of claim 5 or 6, or the degradation agent of claim 7 in the degradation of zearalenone.