Application of GhZH protein in efficient hydrolysis of zearalenone and its metabolic derivatives

Abstract

The present invention belongs to the field of agricultural technology and relates to the degradation of zearalenone, particularly the use of GhZH proteins and encoded genes in the efficient hydrolysis of zearalenone and its metabolic derivatives. The amino acid sequence of the protein GhZH with efficient zearalenone hydrolase activity involved in the present invention is shown in SEQ ID NO.1, and the nucleotide sequence of the gene encoding it is shown in SEQ ID NO.2. The present invention also discloses a method for preparing a biomaterial containing the zearalenone hydrolase GhZH and its application. The zearalenone hydrolase GhZH provided by the present invention has important application prospects in the field of mycotoxin detoxification in feed and food.

Description

Technical Field

[0001] The invention belongs to the technical field of agriculture, relates to the degradation of zearalenone, and particularly refers to the application of GhZH protein in the efficient hydrolysis of zearalenone and its metabolic derivatives. Background Art

[0002] Zearalenone (ZEN), also known as F-2 toxin, is a toxic secondary metabolite primarily produced by Fusarium fungi and one of the most common mycotoxin contaminants in the feed and food supply chain. ZEN is frequently detected in grains such as wheat, barley, corn, and sorghum, as well as their processing by-products. Globally, 45% of grain and feed are contaminated with ZEN, causing significant economic losses. As an estradiol analog, ZEN can cause endocrine disruption and reproductive problems in humans and animals. Long-term consumption of ZEN-contaminated diets in female animals can lead to uterine congestion and enlargement, vulvar redness and swelling, rectal prolapse, fetal malformations, and miscarriage. Long-term consumption of ZEN-contaminated diets in male animals can lead to impaired blood-testis barrier function, decreased libido, and decreased sperm motility and count.

[0003] ZEN also has a variety of metabolic derivatives, mainly including α-zearalenol (α-Zearalenol, α-ZOL), β-zearalenol (β-Zearalenol, β-ZOL), α-zearalanol (α-ZAL) and β-zearalanol (β-ZAL). These metabolic derivatives still have certain toxicity, among which α-ZOL is 30 times more toxic than ZEN.

[0004] Enzymatic hydrolysis can effectively reduce the toxicity of ZEN and its metabolites in feed and food into non-toxic or low-toxic metabolites. Current research on ZEN-degrading enzymes focuses primarily on the fungal lactone hydrolase ZHD101 and its homologous proteins. These ZEN-degrading enzymes generally have an optimal pH of 8.0–9.5. However, the pH of the animal and human intestines ranges from 6.0 to 8.0, resulting in their inability to fully hydrolyze ZEN in these environments. Furthermore, these ZEN-degrading enzymes generally have low activity against the more toxic α-ZOL (Table 1).

[0005] Table 1 Comparison of fungal zearalenone hydrolase ZHD101 family proteins

[0006]

[0007] The existing family of fungal zearalenone hydrolases has an optimal pH at an alkaline level, exhibits low enzymatic activity, and cannot efficiently degrade both ZEN and α-ZOL simultaneously. Therefore, there is an urgent need to explore and exploit more zearalenone hydrolase resources to achieve efficient hydrolysis of ZEN and its derivatives, particularly the more toxic α-ZOL, under neutral conditions. Summary of the Invention

[0008] In order to solve the above technical problems, the present invention proposes an application of GhZH protein in the efficient hydrolysis of zearalenone and its metabolic derivatives.

[0009] The technical solution of the present invention is achieved as follows:

[0010] The amino acid sequence of the GhZH protein provided by the present invention is shown in SEQ ID NO.1:

[0011] The present invention also provides the nucleotide sequence of the GhZH protein encoding gene, as shown in SEQ ID NO.2.

[0012] Furthermore, the present invention also provides a recombinant expression vector comprising a gene encoding the GhZH protein.

[0013] Furthermore, the present invention also provides a recombinant strain expressing GhZH protein, which is obtained by transforming a recombinant expression vector containing a GhZH encoding gene. The recombinant strain can be Escherichia coli, Bacillus subtilis, Bacillus licheniformis, Lactococcus lactis, Lactobacillus plantarum, Lactobacillus casei, Pichia pastoris, Saccharomyces cerevisiae, Kluyveromyces lactis, Aspergillus oryzae, Aspergillus niger and Trichoderma reesei.

[0014] Furthermore, the present invention also provides a method for preparing GhZH protein using Escherichia coli, comprising the following steps:

[0015] 1) Transform Escherichia coli Rosetta (DE3) with the recombinant expression vector pET-31b-GhZH containing the GhZH protein encoding gene to obtain a recombinant strain;

[0016] 2) Cultivating the recombinant strain to induce GhZH protein expression.

[0017] Furthermore, the present invention also provides a biomaterial containing GhZH protein, wherein the biomaterial comprises GhZH protein and a physiologically acceptable carrier thereof, wherein the content of the GhZH protein is 0.01% to 5%, and the physiologically acceptable carrier comprises one or more of chitosan, chitin, starch, sucrose, glucose, oligofructose, rice, rice bran, rice husk powder, soybean hull, wheat bran, corn cob powder, water-soluble starch, maltodextrin and cyclodextrin.

[0018] The biomaterial of the present invention can be used to degrade zearalenone and its derivatives in grains, feed, fruit juice, pomace, nuts, corn steep liquor and Chinese herbal medicine, wherein the zearalenone derivative is any one of α-zearalenol, β-zearalenol, α-zearalanol and β-zearalanol.

[0019] The present invention has the following beneficial effects:

[0020] (1) The bacterial zearalenone hydrolase GhZH provided by the present invention is a novel zearalenone hydrolase that belongs to a different family from previously reported fungal zearalenone hydrolases. The GhZH protein provided by the present invention has an enzymatic activity of 639 U / mg in hydrolyzing ZEN, which is much higher than the enzymatic activity of the previously reported fungal zearalenone hydrolase ZHD101 and its homologous proteins in hydrolyzing ZEN. In addition, GhZH has an enzymatic activity of 582 U / mg against the more toxic ZEN derivative α-ZOL.

[0021] (2) The GhZH protein of the present invention is a neutral zearalenone hydrolase with an optimal reaction pH of 7.0, close to the physiological pH of the animal and human intestines. In addition, biomaterials containing the GhZH protein can effectively degrade ZEN in corn steep liquor and apple juice, showing promising application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0023] Figure 1 The figure shows the SDS-PAGE analysis results of the purified GhZH protein.

[0024] Figure 2 The HPLC analysis results of GhZH protein degradation of ZEN.

[0025] Figure 3 The effect of reaction pH on GhZH enzyme activity. DETAILED DESCRIPTION

[0026] The following will clearly and completely describe the technical solutions of the present invention in conjunction with the embodiments of the present invention. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0027] The main experimental materials and reagents used in the examples of the present invention are:

[0028] The Escherichia coli expression vector pET-31b and Escherichia coli Rosetta (DE3) were purchased from Invitrogen, restriction endonucleases, DNA polymerase, and DNA ligase were purchased from New England Biolabs, and zearalenone, α-zearalenol, β-zearalenol, α-zearalanol, and β-zearalanol were purchased from Sigma.

[0029] Unless otherwise specified, other biochemical reagents used in the examples are commercially available reagents, and the technical means used in the examples are conventional means used by those skilled in the art.

[0030] Example 1: Cloning of the GhZH protein encoding gene

[0031] The hydrophobic Gordonia bacteria ( Gordonia hydrophobica ) were inoculated into 5 mL of LB liquid medium and cultured overnight at 37°C with shaking at 180 rpm. The cells were harvested by centrifugation at 12,000 rpm for 1 minute, and genomic DNA was extracted using the Tiangen Bacterial Genomic DNA Extraction Kit (Cat. No. DP302). The GhZH protein-encoding gene was amplified and cloned using PCR using the extracted genomic DNA as a template using synthetic primers P1 and P2.

[0032] Upstream primer P1 (SEQ ID NO. 3): 5'ATGGCAATTCAACAGGGC3'

[0033] Downstream primer P2 (SEQ ID NO. 4): 5'TTATCGGGCGGCAGGAAG3'

[0034] The PCR reaction system was as follows: 1 μL template DNA, 2 μL upstream primer P1, 2 μL downstream primer P2, 25 μL 2× PfuPCR Mix, and 20 μL ddH2O.

[0035] The amplification conditions were as follows: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 s, annealing at 50°C for 30 s, extension at 72°C for 1 min, 30 cycles; and complete extension at 72°C for 10 min.

[0036] The PCR amplification products were subjected to 1% agarose gel electrophoresis and recovered using Tiangen Company's ordinary agarose gel DNA recovery kit (catalog number DP209) and sent to Shanghai Bioengineering Company for sequencing.

[0037] According to the sequencing results, the full-length sequence of the GhZH protein encoding gene is shown in SEQ ID NO.2; the amino acid sequence of the GhZH protein is shown in SEQ ID NO.1, which contains 322 amino acids and no signal peptide is predicted.

[0038] Example 2: GhZH protein expression and purification

[0039] The coding gene of the GhZH protein cloned in Example 1 was connected between the NdeI and XhoI restriction sites of the pET-31b vector, and the recombinant plasmid pET-31b-GhZH was transformed into Escherichia coli competent cells Rosetta (DE3).

[0040] The recombinant Escherichia coli Rosetta (DE3) transformed with the pET-31b-GhZH plasmid was inoculated into 5 mL of LB liquid medium for activation overnight, and then transferred to a 500 mL Erlenmeyer flask with a liquid volume of 300 mL at a ratio of 1:100. The culture was shaken at 37°C and 180 rpm until the OD600 reached 0.6. 0.4 mM IPTG was added and the culture was shaken at 28°C and 180 rpm for 6 h to induce the expression of the target protein.

[0041] The fermentation broth was collected and centrifuged at 12,000 rpm for 30 minutes at 4°C. The supernatant was discarded. The cells were resuspended in pH 7.0 phosphate buffer and centrifuged at 12,000 rpm for 30 minutes at 4°C. The supernatant was discarded and the cells were washed three times. The cells were resuspended in Binding buffer and disrupted by ultrasonication in an ice-water bath. The cells were centrifuged at 12,000 rpm for 10 minutes at 4°C. The supernatant was collected to obtain the crude GhZH protein.

[0042] The crude GhZH protein was filtered through a 0.45 μm filter membrane and purified by nickel ion affinity chromatography column (Ni 2+ -NTA) to purify the recombinant protein. For equilibration, loading, elution, and other steps, refer to the Qiagen 6xHis-tagged Protein Purification Manual.

[0043] The purified protein was ultrafiltered using a 3 kDa cut-off tube to remove imidazole, and the purified GhZH was analyzed by SDS-PAGE electrophoresis.

[0044] The results are as follows Figure 1 As shown, lane 1 is the migration band of the purified GhZH protein with a molecular weight of approximately 37 kDa, which is consistent with the theoretical molecular weight.

[0045] Example 3: GhZH protein hydrolysis ZEN function test

[0046] Solid ZEN standard was dissolved in methanol to prepare a 1.5 mg / mL stock solution. The experiment was performed using the following 500 μL reaction system: 485 μL sodium phosphate buffer (100 mM, pH 7.0), 5 μL GhZH protein (0.1 mg / mL), and 10 μL ZEN stock solution. The reaction was incubated in a 37°C water bath for 0, 0.5, and 1.0 h, and then terminated by adding 500 μL of methanol. The ZEN content in the reaction system was determined by HPLC.

[0047] The chromatographic conditions for HPLC detection of ZEN were as follows: chromatographic column: Agilent C18 column, 4.6 mm × 250 mm × 5 μm; mobile phase: acetonitrile-water (60:40); flow rate: 1 mL / min; column temperature: 30°C; injection volume: 20 μL; UV detector detection wavelength: 274 nm; acquisition time: 20 minutes.

[0048] Figure 2 Shown are the HPLC analysis results of the GhZH protein ZEN hydrolysis activity test. The ZEN retention time is 7.4 min. After 0.5 h of reaction, the ZEN peak area in the chromatogram decreased significantly, and a hydrolysis product was generated at the retention time of 2.1 min. After 1.0 h of reaction, the ZEN peak area further decreased, and the hydrolysis product peak area further increased. The above results indicate that GhZH protein has the function of hydrolyzing ZEN. According to the peak area calculation, the ZEN degradation rate reached 71% after 0.5 h of reaction, and the ZEN degradation rate reached 93% after 1.0 h of reaction.

[0049] Example 4: Determination of the enzymatic activity of GhZH protein hydrolyzing ZEN and its derivatives

[0050] Solid standards of ZEN, α-ZOL, β-ZOL, α-ZAL, and β-ZAL were dissolved in methanol to prepare a 1.5 mg / mL stock solution. The following 500 μL reaction system was used: 485 μL of sodium phosphate buffer (100 mM, pH 7.0), 5 μL of GhZH protein (0.1 mg / mL), and 10 μL of the ZEN, α-ZOL, β-ZOL, α-ZAL, or β-ZAL stock solution. After incubation at 37°C in a water bath for 10 minutes, the reaction was terminated by adding 500 μL of methanol. The ZEN, α-ZOL, β-ZOL, α-ZAL, or β-ZAL content in the reaction system was determined using the HPLC method described in Example 3. Enzyme activity was calculated based on the amount of substrate degradation. Enzyme activity units (U) are defined as the amount of enzyme required to convert 1 μg of substrate per minute at 37°C.

[0051] Table 2 Enzymatic activity of GhZH protein in hydrolyzing ZEN and its derivatives

[0052]

[0053] The results are shown in Table 2. The enzymatic activities of GhZH protein in hydrolyzing ZEN, α-ZOL, β-ZOL, α-ZAL and β-ZAL were 639 U / mg, 582 U / mg, 531 U / mg, 703 U / mg and 681 U / mg, respectively.

[0054] Example 5: Effect of reaction pH on GhZH enzyme activity

[0055] The following 500 μL reaction system was used: 5 μL of GhZH protein (0.1 mg / mL) and 10 μL of ZEN stock solution were added to 485 μL of buffer at different pH values. After incubation at 37°C in a water bath for 10 minutes, the reaction was terminated by adding 500 μL of methanol. The ZEN content in the reaction system was determined using the HPLC method described in Example 3, and the enzyme activity was calculated based on the amount of substrate degradation. The enzyme activity at the optimal pH was defined as 100%, and the relative enzyme activity at different pH values ​​was calculated.

[0056] The results are as follows Figure 3 As shown in the figure, the optimal reaction pH for GhZH protein hydrolysis of ZEN is 7.0, and its relative enzyme activity is maintained above 64% in the pH range of 6.0-9.0.

[0057] Example 6: Biomaterial containing GhZH protein

[0058] 10 g of a carrier was added to 100 mL of GhZH protein (1 mg / mL). The carrier was prepared by mixing maltodextrin and glucose in a mass ratio of 5:1. The mixture was freeze-dried in a vacuum to obtain a biomaterial containing the GhZH protein.

[0059] 100 mL of corn steep liquor containing 6.2 μg / mL ZEN was added to a 500 mL Erlenmeyer flask, the pH was adjusted to 7.0 with 1 M NaOH, 1.0 g of the biomaterial prepared in Example 6 was added, and the ZEN residue in the corn steep liquor was measured after reaction at 37° C. for 1 h, 2 h, and 4 h.

[0060] 100 mL of apple juice containing 1.2 μg / mL ZEN was added to a 500 mL Erlenmeyer flask, and 1.0 g of the biomaterial prepared in Example 6 was added. The reaction was carried out at 37° C. for 1 h, 2 h, and 4 h, and the ZEN residue in the apple juice was measured.

[0061] The determination method is as follows: take 10 mL of corn syrup or apple juice, add 5 mL of methanol-water mixture (volume ratio 3:2), vortex and oscillate for 10 minutes, then filter through glass fiber filter paper, and purify the filtrate through an immunoaffinity column (product of China Inspection Weikang, product number: IAC105). The ZEN content is detected by the high-performance liquid chromatography analysis method described in Example 3.

[0062] Table 3 Degradation effect of biomaterials containing GhZH protein on ZEN in corn steep liquor

[0063]

[0064] Table 4 Degradation effect of biomaterials containing GhZH protein on ZEN in apple juice

[0065]

[0066] The results of degradation of ZEN in corn steep liquor by biomaterials containing GhZH protein are shown in Table 3. After 1 h, 2 h and 4 h of reaction, the ZEN content in corn steep liquor decreased from 6.2 μg / mL to 3.3 μg / mL, 1.2 μg / mL and 0.1 μg / mL, respectively.

[0067] The results of degradation of ZEN in apple juice by biomaterials containing GhZH protein are shown in Table 4. After 1 h, 2 h and 4 h of reaction, the ZEN content in apple juice decreased from 1.2 μg / mL to 0.9 μg / mL, 0.5 μg / mL and 0.2 μg / mL, respectively.

[0068] These results demonstrate the feasibility of using GhZH protein loaded onto a carrier as a biomaterial for ZEN degradation. Chitosan, chitin, starch, sucrose, glucose, oligofructose, rice, rice bran, rice husk powder, soybean hulls, wheat bran, corncob powder, soluble starch, maltodextrin, and cyclodextrin are commonly used biomaterials, making this feasible. Clearly, in addition to corn steep liquor and apple juice, biomaterials containing GhZH protein can also be applied to remove ZEN and its metabolic derivatives from cereals and feed, pomace, nuts, and Chinese herbal extracts.

[0069] 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 in the scope of protection of the present invention.

Claims

1. Application of GhZH protein in the efficient hydrolysis of zearalenone and its metabolic derivatives, characterized by: The amino acid sequence of the GhZH protein is shown in SEQ ID No. 1; The metabolic derivative is any one of α-zearalenol, β-zearalenol, α-zearalanol and β-zearalanol.

2. The use according to claim 1, characterized in that: The GhZH protein has zearalenone hydrolase activity.

3. The use according to claim 1, characterized in that: The nucleotide sequence encoding the GhZH protein is shown in SEQ ID No.

2.

4. Use of a biomaterial containing GhZH protein for removing zearalenone and its metabolic derivatives from cereals, feed, juice, pomace, nuts, corn steep liquor or Chinese herbal medicine extracts, characterized by: The amino acid sequence of the GhZH protein is shown in SEQ ID No. 1; The metabolic derivative is any one of α-zearalenol, β-zearalenol, α-zearalanol and β-zearalanol.

5. The use according to claim 4, characterized in that: The biological material refers to the expression product of the recombinant vector, wherein the recombinant vector is an expression vector comprising a nucleotide fragment expressing the GhZH protein.

6. The use according to claim 5, characterized in that: The recombinant vector comprises the nucleotide fragment shown in SEQ ID No.

2.

7. The use according to claim 6, characterized in that: The mass fraction of the GhZH protein in the biomaterial is 0.01%-5%.

8. The use according to claim 7, characterized in that: The carrier of the biomaterial is one or more of chitosan, chitin, starch, sucrose, glucose, oligofructose, rice, rice bran, rice husk powder, soybean hull, wheat bran, corn cob powder, water-soluble starch, maltodextrin and cyclodextrin.

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