Use of an enzyme in the degradation of zearalenone
By using an enzyme derived from Xylaria multiplex, with the amino acid sequence shown in SEQ ID NO.1, the problem of difficult removal of zearalenone in the prior art has been solved, achieving a highly efficient and safe biological detoxification effect, which is suitable for the food and feed industries.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- SUZHOU ENZYME BIOTECHNOLOGY CO LTD
- Filing Date
- 2023-05-31
- Publication Date
- 2026-06-05
AI Technical Summary
Existing technologies for removing zearalenone have problems such as high cost, difficult operation, potential environmental pollution and alteration of food properties. Furthermore, microbial detoxification is subject to uncontrollable factors such as decreased activity and nutrient consumption, making it difficult to apply in actual production.
A novel enzyme derived from Xylaria multiplex, with the amino acid sequence shown in SEQ ID NO.1, is used to efficiently catalyze the degradation of zearalenone into non-toxic substances, with a degradation rate of over 90%, making it suitable for biological detoxification in the food and feed industries.
It achieves efficient and safe degradation of zearalenone, maintaining the original nutrition and flavor of food and feed, and has broad application prospects.
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Figure CN117796489B_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of biotechnology and relates to the application of an enzyme in the degradation of zearalenone. Background Technology
[0002] Zearalenone (ZEN) is a toxic compound produced by fungal metabolism, threatening food safety and agricultural production. ZEN is mutagenic, teratogenic, carcinogenic, nephrotoxic, immunotoxic, and genotoxic, and has toxic effects on human and animal health.
[0003] Currently, methods for removing ZEN toxins are mainly divided into physical, chemical, and biological methods. Physical methods include mechanical separation, high-temperature inactivation, radiation treatment, or adsorbents; chemical methods treat the toxins with acidic or alkaline solutions or other compounds. However, methods for removing ZEN through physical and chemical treatments have limitations. For example, heat treatment cannot effectively passivate ZEN; while extrusion and the use of oxidants can reduce ZEN content to some extent, their application in feed and food preparation is limited due to high costs, sample quality loss, and low efficiency and specificity. In summary, physical methods suffer from high costs, operational difficulties, and the potential for product contamination of grains, oils, feed, or the environment; chemical methods may alter feed properties and produce harmful residues, posing food safety issues. These drawbacks limit the application of these two methods in actual production.
[0004] Microbial biocatalysis offers advantages such as mild reaction conditions, environmental friendliness, and sustainability. In existing technologies, CN103937681A discloses a food-grade Aspergillus niger strain and its application in the degradation of zearalenone. This Aspergillus niger was co-cultured with ZEN at a final concentration of 2 ppm for 48 hours under suitable conditions, achieving a ZEN degradation rate of 89.56%. However, microbial detoxification involves many uncontrollable factors in practical operations, such as the introduction of toxic microbial metabolites, decreased activity due to mutations, and the consumption of nutrients in the food during detoxification. CN107217046A discloses a zearalenone toxin-degrading enzyme, ZENdease-N1, its encoding gene, and its applications. This enzyme can efficiently and stably degrade zearalenone and maintains stable zearalenone degradation activity in different environments. Nevertheless, due to insufficient activity or stability, ZEN lactone bond hydrolases reported to date have rarely been applied in actual production.
[0005] In conclusion, the discovery of novel enzymes that degrade zearalenone while preserving the original nutrients and flavor of food or feed is of great significance for the field of biological detoxification of food and feed. Summary of the Invention
[0006] In response to the shortcomings of existing technologies and practical needs, this invention provides an application of an enzyme in the degradation of zearalenone. This invention discovers a novel enzyme for degrading zearalenone, which is expected to be applied to biological detoxification in the food and feed industries, and plays a very important role in the healthy development of agriculture and animal husbandry.
[0007] To achieve the above objectives, the present invention adopts the following technical solution:
[0008] In a first aspect, the present invention provides the application of an enzyme in the degradation of zearalenone, wherein the amino acid sequence of the enzyme comprises:
[0009] (1) The sequence shown in SEQ ID NO.1, or,
[0010] (2) An amino acid sequence obtained by substituting, deleting, or adding one or at least two amino acid residues to the sequence described in (1), and having the same or similar function to the sequence described in (1), or,
[0011] (3) An amino acid sequence that has at least 90% sequence homology with the sequence described in (1) or (2) and has the same or similar function as the sequence described in (1).
[0012] In this invention, an enzyme derived from Xylaria multiplex (amino acid sequence as shown in SEQ ID NO. 1) was discovered for the first time to have the function of degrading zearalenone, catalyzing the degradation of zearalenone into non-toxic substances (reaction as shown in Formula I), thus it is expected to be applied to the degradation of zearalenone, such as biological detoxification in the food and feed industries.
[0013] It is understood that this application has discovered that an enzyme derived from Xylaria multiplex (amino acid sequence as shown in SEQ ID NO. 1) has the function of degrading zearalenone. Therefore, enzymes in Xylaria multiplex or similar strains with the same or similar sequence as the one shown in SEQ ID NO. 1 are expected to have the function of degrading zearalenone. In addition, enzymes obtained by modifying the sequence shown in SEQ ID NO. 1 by substituting, deleting or adding one or more amino acid residues using common techniques in the art, while having the same or similar function as the original protein, are also expected to have the function of degrading zearalenone.
[0014]
[0015] SEQ ID NO.1:
[0016] MRTKGTTTTSEGITWYYEQEGSGPDIVLIPDGLGECHMFDKAIPLIVASGFRVTTFDMPGMSRSSDAPPETFEDVTAQKLASYVISIVDKLGIDTATFWGCSSGGSTVLALVAGYPERVRNALAHEVPTYHI EGLNMLHKLEDEAISTNLAASMPAGSCGNLEAWVGLGGDVHARLWKNYPRWARGYPRTLPLSSPTNASDLTKRPLYWTVGASTSSSRFFDNIVTATKAGVDIGYIPGMHFPYVSDPRAFAQHVVDVTRKHL.
[0017] Secondly, the present invention provides a method for degrading zearalenone, the method comprising:
[0018] The enzyme described in the first aspect is mixed with a sample containing zearalenone degradable to achieve zearalenone degradation.
[0019] In this invention, after co-incubating the enzyme described in the first aspect with zearalenone, a significant decrease in the concentration of zearalenone can be detected, resulting in the production of a corresponding non-toxic product. Ultimately, the degradation rate of zearalenone can reach over 90%.
[0020] Thirdly, the present invention provides the application of the enzyme described in the first aspect in the preparation of zearalenone degradation products.
[0021] Fourthly, the present invention provides a zearalenone biodegradable agent, wherein the biodegradable agent contains the enzyme described in the first aspect.
[0022] Based on the discovered ability to degrade zearalenone, zearalenone-degraded products can be further prepared.
[0023] Preferably, the biodegradable agent further includes any one or a combination of at least two of a carrier, a preservative, or a protein protectant.
[0024] It is understood that carriers, preservatives, or protein protectants commonly used in the field for biodegradable agents are also applicable to this invention.
[0025] Preferably, the carrier comprises a physiologically acceptable compatible carrier.
[0026] Preferably, the physiologically acceptable carrier includes any one or a combination of at least two of the following: rice husk, rice bran, maltodextrin, cyclodextrin, wheat bran, starch, bentonite, oligosaccharides, or yeast cell wall.
[0027] Preferably, the preservative includes any one or a combination of at least two of potassium sorbate, ethylparaben, methylparaben, or antibiotics.
[0028] Preferably, the antibiotic includes any one or a combination of two of penicillin, gentamicin, or vancomycin.
[0029] Preferably, the protein protectant comprises any one or a combination of at least two of bovine serum albumin, mannitol, glycerol, butylene glycol, sodium chloride, or sodium benzoate.
[0030] Compared with the prior art, the present invention has the following beneficial effects:
[0031] This invention is the first to discover an enzyme derived from Xylaria multiplex (amino acid sequence shown in SEQ ID NO.1) that has the function of degrading zearalenone, catalyzing the degradation of zearalenone into non-toxic substances, and thus has the potential to be applied to the degradation of zearalenone, such as biological detoxification in the food and feed industries. Attached Figure Description
[0032] Figure 1 The graph shows the degradation rate of zearalenone in zearalenone.
[0033] Figure 2A This is a graph showing the results of secondary mass spectrometry detection of zearalenone in zearalenone.
[0034] Figure 2B This is a chromatogram of secondary mass spectrometry results for the degradation products of zearalenone in zearalenone.
[0035] Figure 3 The graph shows the degradation rate of toxins in wheat flour.
[0036] Figure 4 The degradation rate of toxins in corn flour. Detailed Implementation
[0037] To further illustrate the technical means and effects of this invention, the following description, in conjunction with embodiments and accompanying drawings, provides a further explanation of the invention. It is understood that the specific embodiments described herein are merely illustrative of the invention and not intended to limit it.
[0038] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.
[0039] Example 1
[0040] This embodiment demonstrates the expression of an enzyme derived from Xylaria multiplex (amino acid sequence shown in SEQ ID NO.1).
[0041] The enzyme gene sequence (SEQ ID NO.2) was synthesized by Shanghai Sangon Biotech Co., Ltd., and the D2P expression plasmid and cell-free expression reaction system (reference: Zhang, K. et al. (2021) Systemic Expression, Purification, and Initial Structural Characterization of Bacteriophage T4 Proteins Without Known Structure Homologs. Front Microbiol 12, 674415.10.3389 / fmicb.2021.674415) were obtained from Shanghai Kangma Biotechnology Co., Ltd. After synthesis, the enzyme sequence was ligated into the protein expression vector plasmid using homologous recombination technology. The recombinant plasmid containing the target gene was used as a reaction template and added to the cell-free reaction system. The reaction was carried out at 37℃ for 3 hours to obtain the target protein. The reaction volume was 1 mL, and the final concentration of the template plasmid was 1 ng / μL. A blank control was set up, i.e., the reaction solution without the template plasmid, to eliminate the influence of enzymes and reaction conditions present in the reaction system.
[0042] SEQ ID NO.2:
[0043] atgagaaccaagggtaccaccaccacctctgaaggtatcacctggtactacgaacaagaaggttctggtccagacatcgttttgatcccagacggtttgggtgaatgtcacatgttcgacaaggctatcccattgatcgttgcttctggtttcagagttaccaccttcgacatgccaggtatgtctagatcttctgacgctccaccagaaaccttcgaagacgttaccgctcaaaagttggcttcttacgttatctctatcgttgacaagttgggtatcgacaccgctaccttctggggttgttcttctggtggttctaccgttttggctttggttgctggttacccagaaagagttagaaacgctttggctcacgaagttccaacctaccacatcgaaggtttgaacatgttgcacaagttggaagacgaagctatctctaccaacttggctgcttctatgccagctggttcttgtggtaacttggaagcctgggttggtttgggtggtgacgttcacgctagattgtggaagaactacccaagatgggctagaggttacccaagaaccttgccattgtcttctccaaccaacgcttctgacttgaccaagagaccattgtactggaccgttggtgcttctacctcttcttctagattcttcgacaacatcgttaccgctaccaaggctggtgttgacatcggttacatcccaggtatgcacttcccatacgtttctgacccaagagctttcgctcaacacgttgttgacgttaccagaaagcacttg。
[0044] Example 2
[0045] This example conducts a test on the degradation of zearalenone (ZEN).
[0046] The enzyme-containing reaction system from Example 1 was used as the experimental group, and the reaction system without template plasmid in Example 1 was used as the blank control. The supernatant of the reaction system was collected by low-speed centrifugation and placed in a new centrifuge tube. OTA (final reaction concentration of 2 μg / mL) was added and incubated at 37℃ for 3 h. After incubation, the system was centrifuged at high speed, and the supernatant was collected. An equal volume of methanol was added, and the system was vigorously shaken and centrifuged at high speed again. The supernatant was filtered through a 0.22 μm organic filter membrane, and the content of ZEN after the reaction was detected based on the liquid chromatography-mass spectrometry method developed by Tian et al. (refer to Azam, MS et al. (2016) Degrading ochratoxin A and zearalenone mycotoxins using a multifunctional recombinant enzyme. Toxins (Basel) 10.3390 / toxins11050301).
[0047] ZEN standards used in the detection experiments were purchased from Sigma-Aldrich, USA. Methanol and acetonitrile used in the experiments were high-performance liquid chromatography (HPLC) grade, manufactured by Merck, Germany. Other HPLC analytical grade solvents and chemicals were provided by Aladdin. Ultrapure water (18.2 M·cm⁻¹) was obtained from Millipore, USA. ZEN detection employed the method developed by Tian et al. The HPLC system was a Thermo Scientific Accela 1250 HPLC system, with an Agilent Extend-C18 column (100 mm × 4.6 mm, 3.5 μm). The column temperature was set to 30 °C, the sample pan temperature to 4 °C, and the sample injection volume to 10 μL. The mobile phase A was 5 mM ammonium acetate aqueous solution, and phase B was pure methanol; the mobile phase flow rate was 0.35 mL / min. The gradient elution program was as follows: 0 min, 15% B; 1 min, 15% B; 6.5 min, 90% B; 8.5 min, 90% B; 9 min, 15% B; 12 min, 15% B. Mass spectrometry analysis was performed using a TSQ Vantage™ triple quadrupole mass spectrometer (Thermo Fisher Scientific, USA) with alternating positive and negative electrospray ionization modes. The nebulizer (N2) and dry gas (N2) pressures were 30 psi and 20 psi, respectively. Ion source parameters were as follows: collision voltage: 3.5 kV (ESI+) and 3.0 kV (ESI-); transport capillary temperature: 250 °C; nebulizer temperature: 350 °C. The concentration of each component in the liquid sample (ug / mL) was calculated as C*F / 1000. Where C is the instrument reading concentration in ng / mL; V is the sample extract volume in mL; M is the total sample weight in mg; and F is the dilution factor. Degradation rate = (M1-M2) / M1*100%. M1 is the ZEN concentration before the reaction, and M2 is the ZEN concentration after the enzyme reaction.
[0048] like Figure 1 As shown, the enzyme derived from Xylaria multiplex, with a length of 263 amino acids, exhibited a degradation rate of 91.07% for ZEN under the experimental conditions, demonstrating good degradation efficiency. Further verification by secondary mass spectrometry confirmed that the product of ZEN degradation catalyzed by the enzyme was the target product. Figure 2A and Figure 2B ).
[0049] Example 3
[0050] In this embodiment, the enzyme-containing reaction system from Example 1 was used as the experimental group, and the reaction system from Example 1 without the template plasmid was used as the blank control. ZEN degradation in wheat flour (Fulinmen wheat core general-purpose wheat flour, purchased from JD.com) and corn flour (Beichun organic corn flour, purchased from JD.com) was carried out respectively.
[0051] The specific experimental procedure includes:
[0052] ZEN standard was added to wheat flour and corn flour at a final concentration of 1000 ng / g. 500 μL of enzyme-containing reaction system was mixed with 500 mg of wheat flour and corn flour with added ZEN, respectively. The same mixing operation was performed on the reaction system without template plasmid as a control. After incubation at 37°C for 3 h, 500 μL of methanol was added to stop the reaction.
[0053] ZEN detection and degradation rate calculation are based on Example 2.
[0054] The results are shown in the figure. The degradation rate of toxins in wheat flour was 69.11%. Figure 3 The degradation rate in corn flour was 39.71%. Figure 4 This indicates that the enzyme discovered in this invention can directly degrade ZEN in food crops when used alone, and has broad application prospects in the development of biodegradable agents.
[0055] In summary, this invention is the first to discover an enzyme derived from Xylaria multiplex (amino acid sequence as shown in SEQ ID NO.1) that has the function of degrading zearalenone, catalyzing the degradation of zearalenone into non-toxic substances, thus showing promise for application in the degradation of zearalenone with a degradation rate of over 90%, and has substantial application value and great significance in the biological detoxification of food and feed.
[0056] The applicant declares that the detailed method of the present invention is illustrated by the above embodiments, but the present invention is not limited to the above detailed method, that is, it does not mean that the present invention must rely on the above detailed method to be implemented. Those skilled in the art should understand that any improvements to the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.
Claims
1. The application of an enzyme in the degradation of zearalenone, characterized in that, The amino acid sequence of the enzyme is as follows: The sequence shown in SEQ ID NO.
1.
2. A method for degrading zearalenone, characterized in that, The method includes: The enzyme described in claim 1 is mixed with a sample containing zearalenone to achieve the degradation of zearalenone.
3. The application of an enzyme in the preparation of zearalenone biodegradable agents, characterized in that, The biodegrading agent contains the enzyme according to claim 1; the amino acid sequence of the enzyme is shown in SEQ ID NO.
1.
4. The application according to claim 3, characterized in that, The biodegradable agent also includes any one or a combination of at least two of the following: a carrier, a preservative, or a protein protectant.
5. The application according to claim 4, characterized in that, The carriers include physiologically acceptable compatible carriers.
6. The application according to claim 5, characterized in that, Physiologically acceptable compatible carriers include any one or a combination of at least two of the following: rice husk, rice bran, maltodextrin, cyclodextrin, wheat bran, starch, bentonite, oligosaccharides, or yeast cell walls.
7. The application according to claim 4, characterized in that, The preservative includes any one or a combination of at least two of potassium sorbate, ethylparaben, or methylparaben.
8. The application according to claim 4, characterized in that, The protein protectant includes any one or a combination of at least two of bovine serum albumin, mannitol, glycerol, butylene glycol, sodium chloride, or sodium benzoate.