Zearalenone degrading enzyme and application thereof
By mining and heterologously expressing zearalenone-degrading enzymes from marine microorganisms, the problems of limited enzyme types and poor stability in existing technologies have been solved, achieving highly efficient zearalenone degradation, which is suitable for the food and feed industries.
Patent Information
- Application Number
- CN202511606735.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-11-05
- Publication Date
- 2026-01-20
AI Technical Summary
In the existing technology, there are few types of zearalenone-degrading enzymes and they are unstable, which limits their application in food and feed, and poses problems such as food safety risks and low degradation efficiency.
Novel zearalenone-degrading enzyme genes were discovered from marine microbial genome databases, heterologously expressed, and recombinant expression vectors were constructed and transformed into host bacteria to obtain recombinant engineered bacteria. Zearalenone-degrading enzymes were obtained through induced culture and used to prepare degrading agents.
The novel zearalenone-degrading enzyme achieves a degradation rate of up to 62.79% for 20 µg/ml zearalenone within 10 minutes, and maintains its activity within the range of 10–50 °C and pH 4.0–9.0, making it suitable for grain storage, food processing, and feed preparation.
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Abstract
Description
TECHNICAL FIELD
[0001] The application belongs to the technical field of zearalenone degrading enzyme, and particularly relates to a zearalenone degrading enzyme and application thereof. BACKGROUND
[0002] Zearalenone (ZEN) is a non-steroidal estrogenic mycotoxin produced mainly by Fusarium. Its molecular formula is C 18 H 22 O5, has a lactone structure, and has a relative molecular weight of 318 and thermal stability. ZEN widely pollutes cereals such as wheat and corn. The estrogenic effect of ZEN has a serious toxic effect on humans and animals. After ingestion, it can cause a decrease in estrogen levels and interfere with normal reproductive function. Reports show that zearalenone has reproductive toxicity, immunotoxicity, genetic toxicity and cytotoxicity. Therefore, it is of great significance to find a safe and effective method to degrade it into a low-toxic or non-toxic substance.
[0003] The conventional detoxification methods of zearalenone include physical detoxification (physical classification, high-temperature treatment, adsorption method and radiation method, etc.) and chemical detoxification (alkali, salt, oxidizing agent, ozone treatment, etc.). Although it has a certain detoxification effect, it has the problems of poor safety, high cost, harsh conditions, environmental pollution, etc. Microbial detoxification method, i.e. using microorganisms and enzymes produced by microorganisms to degrade ZEN, has the advantages of safety, high efficiency, strong specificity, environmental friendliness, etc., and has become a research hotspot. According to reports, Lactobacillus paracasei ZEN can be degraded into α-ZOL and β-ZOL. Rhodococcus erythropolis The degradation rate of NI1 to 1 mg / L ZEN is as high as 84.76%. Since the strains have different metabolic networks and the metabolic mechanisms of most strains are not clear, if they are directly introduced into food to degrade ZEN, there are potential risks of destroying food flavor and threatening food safety. Therefore, researchers try to mine and heterologously express ZEN degrading enzymes, and some achievements have been made. For example, Kosawang et al. cloned zhd101 gene from Clonostachys rosea , and heterologously expressed it. The results showed that ZHD101 hydrolase showed significant degradation effect on ZEN. Yu et al. isolated lactone hydrolase ZHD607 from Phialophora americana , which showed an enzyme activity of 4940 ± 28.06 U / mg under the conditions of pH 8.0 and 35℃ for 30 min. Zhang et al. also identified ZEN degrading enzyme ZENG from Gliocladium roseum , and its hydrolysis products are α-zearalenol (α-ZOL) and α-zearalanol (α-ZAL).
[0004] The degradation of ZEN toxin by enzymes produced by microorganisms is a key method to solve the problem of its pollution of grain. So far, although some research results have been achieved, the ZEN degrading enzyme genes successfully isolated and heterologously expressed are not many, and the ones with stable and efficient degradation effect are even fewer, thereby greatly reducing its application in actual systems. SUMMARY
[0005] The present application aims at providing a zearalenone degrading enzyme and its application. Based on computer-aided technology, a novel ZEN hydrolytic enzyme is mined from a marine microbial genome database, and is heterologously expressed, thereby expanding the types of ZEN degrading enzymes and meeting the market demand for ZEN degrading enzyme preparations, and identifying the basis for the research on the biological detoxification of ZEN in food-grade feed.
[0006] TECHNICAL SOLUTION In a first aspect, the present application provides a zearalenone degrading enzyme, wherein the amino acid sequence of the zearalenone degrading enzyme is shown as SEQ ID NO: 2.
[0007] In a second aspect, the present application provides a coding gene of a zearalenone degrading enzyme, wherein the nucleotide sequence of the coding gene is shown as SEQ ID NO: 1.
[0008] In a third aspect, the present application provides a recombinant expression vector comprising the above-mentioned coding gene.
[0009] In a fourth aspect, the present application provides a recombinant engineering bacterium comprising the above-mentioned coding gene or comprising the above-mentioned recombinant expression vector.
[0010] In a fifth aspect, the present application provides the above-mentioned coding gene, the above-mentioned recombinant expression vector, or the above-mentioned recombinant engineering bacterium for use in the preparation of a zearalenone degrading enzyme, wherein the amino acid sequence of the zearalenone degrading enzyme is shown as SEQ ID NO: 2.
[0011] As a specific embodiment, the use comprises constructing a recombinant expression vector containing the coding gene, transforming the recombinant expression vector into a host bacterium, obtaining a recombinant bacterium, and inducing the culture of the recombinant bacterium, so as to obtain the zearalenone degrading enzyme.
[0012] In a sixth aspect, the present application provides the above-mentioned zearalenone degrading enzyme, the above-mentioned coding gene, the above-mentioned recombinant expression vector, or the above-mentioned recombinant engineering bacterium for use in the degradation of zearalenone.
[0013] In a seventh aspect, the present application provides use of the zearalenone degrading enzyme, the coding gene, the recombinant expression vector or the recombinant engineering bacteria in the preparation of a zearalenone degrading agent.
[0014] In an eighth aspect, the present application provides a zearalenone degrading agent, which comprises the zearalenone degrading enzyme.
[0015] In a ninth aspect, the present application provides a zearalenone degrading method, which comprises the step of mixing the zearalenone degrading enzyme or the degrading agent with zearalenone.
[0016] Advantages: Compared with the prior art, the present application provides a novel zearalenone degrading enzyme OC4 gene sequence, amino acid sequence and expression system derived from marine microorganisms, and verifies the application of the enzyme in zearalenone degradation. Experimental research shows that the zearalenone degrading enzyme OC4 of the present application has a degradation rate of up to 62.79% within 10 min for a final concentration of 20 µg / ml ZEN, showing a high degradation efficiency. ZHD-OC4 can maintain activity within the temperature and pH range of 10-50℃ and pH 4.0-9.0, showing high stability to the environment. Therefore, the enzyme has high application potential in subsequent zearalenone degradation in the fields of grain storage, food processing and feed preparation. BRIEF DESCRIPTION OF DRAWINGS
[0017] Figure 1 SDS-PAGE analysis of ZHD-OC1-4 expression, wherein: 1, 2 are the supernatant and the resuspended material of the precipitate of the control group, 3-4, 5-6, 7-8, 9-10, 11-12 are the expression of the supernatant and the resuspended material of the precipitate of ZHD101, ZHD-OC1, ZHD-OC2, ZHD-OC3 and ZHD-OC4, respectively.
[0018] Figure 2 ZEN degradation rate of ZHD-OC4 before and after purification.
[0019] Figure 3 Effect of temperature on ZHD-OC4 enzyme activity.
[0020] Figure 4 Effect of pH on ZHD-OC4 enzyme activity. DETAILED DESCRIPTION
[0021] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.
[0022] Example 1: Discovery of a novel zearalenone-degrading enzyme Amino acid sequences of ZHDs reported to have ZEN degradation activity were collected from the literature and subjected to conserved sequence analysis. Based on primary structure, candidate sequences were screened, resulting in 24 sequences, named ZHD-1 to ZHD-24. Enzyme similarity analysis, phylogenetic analysis, sequence and structural similarity analysis, molecular docking and binding efficiency analysis, and prediction of catalytic constants for candidate ZHDs were then performed. Finally, potential novel ZHDs were comprehensively screened, ultimately identifying four promising ZHD sequences: ZHD-2, ZHD-4, ZHD-5, and ZHD-19, named ZHD-OC1, ZHD-OC2, ZHD-OC3, and ZHD-OC4, respectively.
[0023] The amino acid sequence of ZHD-OC4 is shown in SEQ ID NO:2, and the nucleotide sequence of its encoding gene is shown in SEQ ID NO:1.
[0024] Example 2: Heterologous expression and activity verification of novel ZHD
[0025] 1.1 Activation of microbial strains strain E. coli DH5α / pET-22b was removed from the -80°C freezer, thawed, and inoculated into fresh LB liquid medium at a 1% (v / v) inoculation rate. It was incubated overnight at 37°C and 200 rpm in a shaker. An appropriate amount of culture was spread evenly onto the corresponding antibiotic-containing plate and incubated overnight at 37°C. Single colonies were picked from the plate and inoculated into LB medium containing the antibiotic (Amp: 50 μg / mL), and incubated at 37°C and 200 rpm in a shaker until the logarithmic growth phase. The pET22b(+) expression plasmid was extracted using the Solarbio plasmid mini-extraction kit.
[0026] 1.2 Construction of recombinant plasmids The ZHD-OC1~4 corresponding nucleotide sequence was sent to Anhui General Biotechnology Co., Ltd. for synthesis, and the synthesized target gene had NdeI and XhoI restriction sites. The pET22b(+) expression plasmid was double-digested with restriction endonuclease NdeI and XhoI in a 37°C metal bath, and the enzyme digestion system is shown in Table 2-1. The enzyme digestion products were verified by DNA gel electrophoresis and gel recovery for purification of the linearized vector pET22b(+). The concentrations of the recovered and purified target fragment and linear vector were measured using a microspectrophotometer, and the double-digestion ligation system was configured at a vector molar concentration to target fragment molar concentration ratio range of 1:3-1:6 (Table 2-2), and linked by T4 DNA ligase overnight at 16°C to construct the pET-22b-ZHD-OC1~4 recombinant plasmid.
[0027] Table 2-1 Double enzyme digestion system of fragment and plasmid
[0028] Table 2-2 Ligation system of target fragment and vector
[0029] 1.3 Transformation of recombinant plasmid into competent cells This experiment used heat shock method for transformation, and the steps were as follows: (1) Thaw the -80℃ stored competent cells on ice E. coli BL21(DE3); (2) Take 10-15 μL of ligation system or purified recombinant plasmid and gently mix with competent cells, and ice bath for 30 min; (3) Mix the competent cells after mixing at 42°C for 90 s; (4) Ice bath for 2 min; (5) Mix 800 μL of LB medium with the competent cells in a clean bench, and incubate at 37°C, 200 rpm for 1 h; (6) Centrifuge the cultured bacterial solution at 12000 rpm for 1 min, remove the supernatant, and mix again; (7) Spread the bacterial solution on Amp-resistant LB plates and incubate in a 37°C incubator overnight.
[0030] 1.4 Colony PCR verification and sequencing verification A single positive colony on the plated agar was picked with a sterile swab and streaked onto an antibiotic-containing plate and incubated overnight at 37°C. A single colony was picked as a template and a PCR reaction was prepared (Table 2-3). The PCR program for amplifying the target fragment is shown in Table 2-4, and the PCR product was detected by DNA gel electrophoresis. The colony with the correct band size was transferred to LB liquid medium for culture. 3-5 ml of the overnight culture was used to extract plasmid and sent to Beijing Genki Biological Technology Co., Ltd. for sequencing to verify the sequence of the target fragment connected to the recombinant plasmid.
[0031] Table 2-3 PCR reaction system for colony
[0032] Table 2-4 PCR reaction system for fragment amplification
[0033] 1.5 Preservation of bacterial strains The strain with the correct sequence was preserved. The preservation method was as follows: 800 μL of 30% glycerol was added to a cryotube, then 800 μL of bacterial liquid cultured to the logarithmic growth phase was added, the cryotube was tightly capped and inverted to mix evenly, a sealing film was used to seal, and the cryotube was labeled and stored in a -80°C refrigerator for standby use.
[0034] 1.6 Induction of expression (1) The preserved bacterial strain was activated according to the method in 1.1 to serve as a seed culture.
[0035] (2) The seed liquid was transferred to 50 mL of LB liquid medium containing 50 μg / mL Amp at an inoculation amount of 1%, and incubated at 37°C in a 200 rpm shaker until the OD600 reached 0.6-0.8.
[0036] (3) 1M IPTG solution was added to a final concentration of 0.6 mM for protein induction, and the culture was incubated overnight at 20°C in a 200 rpm shaker.
[0037] (4) The bacterial liquid after induction was centrifuged in a centrifuge tube at 4°C and 10000 rpm for 5 min to collect the bacterial cells. Then the bacterial cells were resuspended with 10 mM PBS buffer, centrifuged, and the supernatant was removed to wash the bacterial cells twice to remove impurities.
[0038] (5) Cell disruption: The bacterial cells were resuspended with 3 mL of 10 mM PBS buffer and sonicated in an ice water bath. The disruption conditions were as follows: total duration 10 min, 30% power, 5 s interval after each run. After completion, the cell lysate was centrifuged at 4°C and 12000 rpm for 20 min, and the supernatant after centrifugation was the crude protein.
[0039] 1.7 Protein purification The buffer formula used in the protein purification process is shown in the following table: Table 2-5 Buffer formula for protein purification
[0040] The purification steps are as follows: (1) Take out the nickel column stored in the 4°C refrigerator, place it vertically, and naturally empty the 20% ethanol solution in the column; (2) Add five times the column volume of Bufer A to equilibrate the nickel column; (3) Add the crude protein solution obtained by crushing; (4) Add five times the column volume of Buffer A to elute impurities and 3 mL of Buffer B to elute the target protein, thereby obtaining a protein purification solution; (5) Add five times the column volume of Buffer B and five times the column volume of Buffer A to rinse the nickel column; (6) Store the nickel column using 20% ethanol solution and store it in the 4°C refrigerator; (7) After nickel column purification, use an ultrafiltration centrifuge tube corresponding to the molecular weight to remove salt ions. The specific method is as follows: place the protein purification solution obtained after nickel column purification in an ultrafiltration centrifuge tube, centrifuge at 4°C and 6000 rpm for 30 min, then add ultrapure water to the same scale line in the ultrafiltration centrifuge tube, repeat 2-3 times.
[0041] (8) Take a small amount of protein solution and use the BCA protein concentration determination kit to detect the protein concentration.
[0042] 1.8 SDS-PAGE analysis The cell lysate, cell lysate supernatant, and cell lysate resuspension of the expression strain E. coli BL21(DE3) / pET-22b-ZHD-OC1~4 were subjected to SDS-PAGE electrophoresis detection to observe whether the target protein was expressed in a soluble manner and to explore the effects of induction conditions on protein expression. The specific steps are as follows: (1) Protein gel preparation: assemble the mold and prepare the corresponding concentration of separation gel according to the instructions of the SDS-PAGE gel preparation kit. Pour the separation gel into the glass plate 2 / 3, add water to seal the separation gel, after the separation gel is solidified, absorb the upper layer of water, prepare 5% concentrated gel, immediately pour into the upper layer of the separation gel, insert the comb, and after the concentrated gel is completely solidified, wrap it and store it in the 4°C refrigerator.
[0043] (2) Protein sample treatment: ①According to the loading amount of 80-100 μg of supernatant, 40-50 μg of precipitate, and 10-12 μg of purified protein, the target protein was diluted to an appropriate concentration; ②5 μL of 4×Protein SDS PAGE loading Buffer was added to 15 μL of the target protein, and heating was performed at 100 ℃ for 5 min; ③Then, slow cooling was performed to 60 ℃, and then the sample was placed on ice for complete cooling.
[0044] (3) SDS-PAGE analysis: The protein gel plate was installed into the electrophoresis device, 1×protein running buffer was poured, whether the liquid was leaked was checked, and the comb was pulled out. A protein Marker with a suitable range was selected for spotting, the volume of the Marker and the protein sample was 5 μL, and electrophoresis was performed under the condition of a voltage of 120 V until the eosin blue reached the bottom of the glass plate. The protein gel was peeled off, placed in a protein staining solution, and oscillated for staining at 37 ℃ for 30 min. Finally, oscillation was performed in a protein decolorizing solution, and finally a gel imaging system was used for analysis.
[0045] 1.9 Potential new ZHD crude enzyme activity determination 500 μL of the reaction system contained 10 μL of 1 mg / mL ZEN solution (final concentration 20 ppm), 10 μL of supernatant after induction and crushing, and 480 μL of buffer. The reaction was performed at 35 ℃ for 10 min, and then 500 μL of methanol was added to terminate the reaction. After the termination reaction mixture was filtered through a 0.22 μm filter membrane, the residual amount of ZEN was detected by HPLC.
[0046] The HPLC detection conditions were set as follows: Table 2-6 HPLC detection parameters
[0047] 1.10 Characterization of the properties of the new ZHD enzyme Determination of the optimum temperature: the purified enzyme solution obtained in 2.1.4 was reacted under the reaction conditions of 2.1.6 at temperatures of 10, 20, 30, 40, 50, 60, and 70 ℃, respectively. After the reaction was completed, the residual concentration of ZEN in the system was determined, and the relative enzyme activity was calculated. All experiments were set in triplicate, and the results were averaged.
[0048] Determination of the optimum pH: the purified enzyme solution obtained in 2.1.4 was reacted under the reaction conditions of 2.1.6 at pH values of 3, 4, 5, 6 (citric acid-sodium citrate buffer), 7 (phosphate buffer), 8, 9, and 10 (glycine-sodium hydroxide buffer). The residual concentration of ZEN in the system was determined, and the relative enzyme activity was calculated. All experiments were set in triplicate, and the results were averaged.
[0049] 2.1 SDS-PAGE analysis of ZHD-OC1~4 Take engineered strains E. coli BL21(DE3) ZHD-OC1~4 were cultured in shake flasks and induced to express, followed by disruption and centrifugation to obtain supernatant and precipitate. The precipitate was resuspended in 3 mL PBS, and the supernatant and precipitate were analyzed by SDS-PAGE. Figure 1 The target bands were largely consistent with the theoretical values of ZHD-OC1: 28.2 kDa, ZHD-OC2: 29.8 kDa, ZHD-OC3: 29.8 kDa, and ZHD-OC4: 29.4 kDa. The bands corresponding to ZHD-OC1~3 appeared in the precipitate fraction, indicating that they may have misfolded to form inclusion bodies; the band corresponding to ZHD-OC4 appeared in the lysed supernatant, suggesting that the enzyme folded correctly and was successfully expressed in soluble form. Therefore, ZHD-OC4 was prioritized for purification and enzymatic activity assays.
[0050] 2.2 ZHD-OC4 Activity Verification To verify whether the candidate enzymes truly possess the ability to degrade ZEN, this study first selected ZHD-OC4, which performed well in soluble expression, for crude enzyme activity identification. The lysed supernatant was directly applied to the substrate ZEN, and the degradation effect was preliminarily detected by high-performance liquid chromatography (HPLC). The results showed that the degradation of ZEN was relatively weak, suggesting that the content of soluble proteins in the crude enzyme solution was limited or that there was interference from other proteins.
[0051] ZHD-OC4 was subsequently purified by affinity chromatography and quantitatively analyzed by HPLC. In a reaction system with a substrate concentration of 20 μg / mL ZEN, ZHD-OC4 significantly degraded ZEN, achieving a degradation rate of 62.79%, indicating that the enzyme possesses certain catalytic activity after purification. Figure 2 ).
[0052] 2.3 Optimal temperature of ZHD-OC4 like Figure 3As shown, ZHD-OC4 and ZEN were co-treated with PBS buffer at different temperatures for 1 hour, and the remaining ZEN concentration was determined by HPLC to calculate enzyme activity. At a reaction temperature of 10 °C, the relative enzyme activity was 37.4%, with the highest relative activity at 30 °C. Above 30 °C, the enzyme activity of ZHD-OC4 decreased rapidly with increasing temperature, reaching only 26.1% relative activity at 50 °C. At 60 °C, there was no significant degradation effect, and the relative enzyme activity dropped to 0. Therefore, the optimal reaction temperature for ZHD-OC4 is 30 °C, and it can maintain activity within a relatively wide temperature range of 10–50 °C. However, it is quite sensitive to temperature, and its activity is significantly affected by the reaction temperature.
[0053] 2.4 Optimal pH of ZHD-OC4 like Figure 4 As shown, ZHD-OC4 and ZEN were co-treated at 30℃ in buffer solutions of different pH for 1 h. The remaining ZEN concentration was then determined by HPLC to calculate enzyme activity. ZHD-OC4 exhibited the highest relative enzyme activity at pH 8. When the pH reached 4, the enzyme activity began to decrease rapidly, remaining at less than 60%. When the pH continued to decrease to 3, degradation activity was almost undetectable, and the enzyme activity approached 0. At pH 10, the enzyme activity decreased to less than 20%. The results indicate that ZHD-OC4 has a better degradation effect on ZEN in a slightly alkaline environment, while excessively acidic and alkaline conditions have a significant impact on enzyme activity.
[0054] The embodiments of the present invention have been described in detail above with reference to specific examples. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention.
Claims
1. A zearalenone-degrading enzyme, characterized in that, The amino acid sequence of the zearalenone degrading enzyme is shown as SEQ ID NO:
2.
2. A gene encoding a zearalenone-degrading enzyme, characterized in that, The nucleotide sequence of the coding gene is shown as SEQ ID NO:
1.
3. A recombinant expression vector, characterized in that, The recombinant expression vector comprises the coding gene according to claim 2.
4. A recombinant engineered bacterium, characterized in that, The recombinant engineering bacteria comprise the coding gene according to claim 2, or comprise the recombinant expression vector according to claim 3.
5. The use of the coding gene of claim 2, the recombinant expression vector of claim 3, or the recombinant engineering bacteria of claim 4 in the preparation of zearalenone degrading enzyme, characterized in that, The amino acid sequence of the zearalenone degrading enzyme is shown as SEQ ID NO:
2.
6. Use according to claim 5, characterized in that, The application comprises constructing a recombinant expression vector containing the coding gene, transforming the recombinant expression vector into host bacteria, obtaining recombinant bacteria, and inducing culture of the recombinant bacteria, thereby obtaining the zearalenone degrading enzyme.
7. Use of the zearalenone degrading enzyme according to claim 1, the coding gene according to claim 2, the recombinant expression vector according to claim 3, or the recombinant engineering bacteria according to claim 4 in degrading zearalenone.
8. Use of the zearalenone degrading enzyme according to claim 1, the coding gene according to claim 2, the recombinant expression vector according to claim 3, or the recombinant engineering bacteria according to claim 4 in preparing a zearalenone degrading agent.
9. A zearalenone degrading agent, characterized by comprising: The degrading agent comprises the zearalenone degrading enzyme according to claim 1.
10. A method of degrading zearalenone, characterized by, The method comprises the step of mixing and reacting the zearalenone degrading enzyme according to claim 1 or the degrading agent according to claim 9 with zearalenone.