A neutral zearalenone hydrolase HaZHD and its encoding gene and application

Abstract

The present invention discloses a neutral zearalenone hydrolase HaZHD and its encoding gene and application, which belong to the field of agricultural biotechnology; the amino acid sequence of the zearalenone hydrolase HaZHD involved in the present invention is shown in SEQ ID NO.1, and the nucleotide sequence of its encoding gene hazhd is shown in SEQ ID NO.2, and the above-mentioned hydrolase HaZHD can be used for hydrolyzing zearalenone and cryptic zearalenone; the recombinant expression vector of the zearalenone hydrolase HaZHD involved in the present invention is preferably pET‑31b‑hazhd and pPICZαA‑hazhd, and the recombinant strain is preferably Escherichia coli and Pichia pastoris; the present invention also discloses an additive containing the above-mentioned zearalenone hydrolase HaZHD and its application. The zearalenone hydrolase HaZHD provided by the present invention can simultaneously hydrolyze zearalenone and cryptic zearalenone, and has important application prospects in the field of mycotoxin detoxification in feed and food.

Description

Technical Field

[0001] The invention belongs to the field of agricultural biotechnology, and particularly relates to a neutral zearalenone hydrolase HaZHD, a coding gene thereof and an application thereof. Background Art

[0002] Zearalenone (ZEN), also known as F-2 toxin, is a toxic secondary metabolite primarily produced by Fusarium fungi, such as Fusarium graminearum and Fusarium moniliforme. ZEN is an estrogen analog that can cause endocrine and reproductive disorders in animals and humans. ZEN is also hepatotoxic, immunotoxic, and enterotoxic. ZEN contamination in feed can also leave residues in meat, eggs, and milk, raising animal-derived food safety concerns. Globally, 45% of feed and feed ingredients are contaminated with ZEN, causing significant economic losses to the feed industry and livestock and poultry farming. Recent studies have revealed that ZEN can also exist in cereals in modified forms such as sulfated, glycosylated, and glutathionylated forms, termed "cryptic zearalenone." Common cryptic zearalenone forms include zearalenone-14-sulfate (ZEN-14-S), zearalenone-16-sulfate (ZEN-16-S), zearalenone-14-glucoside (ZEN-14-G), and zearalenone-16-glucoside (ZEN-16-G). Cryptogenic zearalenone is non-toxic or has minimal toxicity itself, but can be converted to zearalenone by microorganisms in the digestive tracts of humans and animals, leading to toxicity.

[0003] Cereals can be infected with toxin-producing molds at all stages of growth, harvesting, and storage. The occurrence of ZEN contamination can be reduced to a certain extent by selecting mold-resistant crop varieties, implementing appropriate field management measures, strictly controlling moisture content and ambient temperature and humidity during harvest and storage, and adding mold inhibitors. Common detoxification methods for cereals and feeds already contaminated with ZEN can be categorized as physical, chemical, and biological. Physical methods can be further divided into two categories: adding organic or inorganic adsorbents to cereals and feeds, and irradiation, microwave heating, and plasma treatment. Chemical methods include ozone fumigation, ammonia treatment, acidification, and electrolytic oxidized water immersion. Biological methods utilize specific microorganisms and the enzymes they produce to catalyze the conversion of ZEN into non-toxic or low-toxic metabolites. Compared to physical and chemical methods, biodegradation offers advantages such as safety, efficiency, environmental friendliness, minimal impact on the nutritional value and sensory quality of feed and food, and ease of scalability. Therefore, it is considered the most promising method for ZEN detoxification.

[0004] Currently, several enzymes have been publicly reported to be capable of degrading ZEN, including the laccase CotA from Bacillus licheniformis, the dye-decolorizing peroxidase DyP from Bacillus subtilis, and the lactone hydrolase ZHD101 from Gliocladium roseum. The ZEN hydrolase ZHD101, as well as other recently reported ZEN hydrolases such as Zhd11B, CbZHD, ZENC, Zhd518, and ZHD607, have an optimal reaction pH between 8.0 and 10.0. However, the pH of the animal and human intestines ranges from 6.0 to 8.0, resulting in low ZEN hydrolysis activity in these environments. Furthermore, whether these ZEN oxidases and hydrolases can simultaneously degrade cryptic ZEN remains understudied.

[0005] Therefore, there is an urgent need for a zearalenone hydrolase that can efficiently hydrolyze ZEN and hidden ZEN at the same time. Summary of the Invention

[0006] The purpose of the present invention is to provide a neutral zearalenone hydrolase HaZHD and its encoding gene and application. The neutral zearalenone hydrolase HaZHD can simultaneously and efficiently hydrolyze zearalenone and hidden zearalenone.

[0007] To achieve the above object, the present invention adopts the following technical solution: a neutral zearalenone hydrolase HaZHD, the amino acid sequence of the zearalenone hydrolase HaZHD is shown in SEQ ID NO.1.

[0008] SEQ ID NO.1:

[0009] MRTKGTITTHEGITWYYEQEGSGPDIVLIPDGLGECQMFDKPMSLIAAEGFRATTFDMPGMSRSSDAPPETYEDVTGQKLASYVISIIDKLGIDTATFWGCSSGGSTVLALVAGYPARVRTALAHEVPTYH IDDLSHLHKLEDEVISANLSATVPAGSCCGDLEAWAGLGDDAHARLWKNYARWAHGYPRTIPQSAATNVEDLLKRPLYWTVGASTPTSRFFDNIVTATKAGVDIGHIPGMHFPYVSHPEAFTKHVVDATRTHL

[0010] The amino acid sequence of the zearalenone hydrolase HaZHD also includes an amino acid sequence that has at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO.1 and has the function of ZEN hydrolase.

[0011] The nucleotide sequence of the gene hazhd encoding the zearalenone hydrolase HaZHD is shown in SEQ ID NO.2.

[0012] SEQ ID NO.2:

[0013] ATGAGGACAAAAGGAACGATAACTACACACGAAGGTATCACCTGGTATTACGAGCAAGAGGGCTCTGGCCCGGATATCGTTCTGATTCCGGATGGTCTGGGTGAATGTCAGATGTTCGACAAGCCGATGTCCCTGATTGCAGCAGAGGGCTTTCGTGCTACGACCTTTGACATGCCGGGTATGTCCAGAAGCAGCGATGCCCCACCGGAAACCTACGAGGACGTTACCGGTCAGAAGTTAGCGAGCTATGTCATCTCGATCATTGACAAACTGGGCATCGACACCGCCACCTTCTGGGGTTGCAGCTCCGGCGGTTCCACCGTGCTGGCCCTGGTCGCAGGCTACCCGGCGCGTGTTCGTACGGCGCTGGCGCATGAAGTGCCAACGTACCATATCGATGACTTGTCTCACCTGCACAAATTGGAGGACGAGGTGATTTCTGCTAATCTGAGCGCGACTGTGCCGGCTGGCAGTTGCGGTGATCTTGAGGCTTGGGCGGGTCTCGGCGACGATGCGCACGCGCGTCTGTGGAAGAACTATGCACGCTGGGCACACGGCTACCCGCGTACCATTCCGCAAAGCGCGGCGACCAACGTGGAAGATTTGTTGAAACGCCCTCTGTATTGGACCGTAGGTGCTAGCACCCCGACAAGCCGCTTCTTCGACAACATTGTGACGGCCACCAAAGCGGGTGTTGACATCGGCCATATCCCGGGTATGCACTTTCCGTATGTTAGCCATCCGGAAGCCTTTACCAAGCACGTTGTTGATGCGACCCGTACCCATTTGTAA

[0014] Neutral zearalenone hydrolase HaZHD is used to hydrolyze zearalenone and masked zearalenone, including zearalenone-14-sulfate, namely "Zearalenone-14-Sulfate, ZEN-14-S", zearalenone-16-sulfate, namely "Zearalenone-16-Sulfate, ZEN-16-S", zearalenone-14-glucoside, namely "Zearalenone-14-Glucoside, ZEN-14-G" and zearalenone-16-glucoside, namely "Zearalenone-16-Glucoside, ZEN-16-G".

[0015] The present invention also provides a recombinant expression vector of a neutral zearalenone hydrolase HaZHD encoding gene, wherein the recombinant expression vector comprises the zearalenone hydrolase HaZHD encoding gene, and the recombinant expression vector is preferably pET-31b-hazhd and pPICZαA-hazhd.

[0016] The present invention also provides a recombinant strain expressing a gene encoding a neutral zearalenone hydrolase HaZHD. The recombinant strain is obtained by transforming a recombinant expression vector encoding the zearalenone hydrolase HaZHD gene. The recombinant strain includes 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.

[0017] Preferably, the recombinant strains are Escherichia coli and Pichia pastoris.

[0018] The present invention also provides a method for preparing zearalenone hydrolase HaZHD using Escherichia coli, comprising the following steps:

[0019] 1) The recombinant expression vector pET-31b-hazhd containing the gene encoding the zearalenone hydrolase HaZHD, hazhd, was used to transform Escherichia coli Rosetta (DE3) to obtain a recombinant strain;

[0020] 2) Cultivating the recombinant strain to induce the expression of zearalenone hydrolase HaZHD.

[0021] The present invention also provides a method for preparing zearalenone hydrolase HaZHD using Pichia pastoris, comprising the following steps:

[0022] 1) Transform Pichia pastoris X33 with the recombinant expression vector pPICZαA-hazhd containing the gene encoding zearalenone hydrolase (HaZHD) to obtain a recombinant strain;

[0023] 2) Cultivating the recombinant strain to induce the expression of zearalenone hydrolase HaZHD.

[0024] The present invention also provides an additive for degrading zearalenone and masked zearalenone, the additive comprising zearalenone hydrolase HaZHD and a physiologically acceptable carrier thereof, wherein the content of the zearalenone hydrolase HaZHD is 0.01%-10%, and the physiologically acceptable carrier comprises one or more of limestone, talc, bentonite, kaolin, montmorillonite, chitosan, chitin, starch, sucrose, rice, rice bran, rice husk powder, soybean hull, wheat bran, corn cob powder, maltodextrin, cyclodextrin and yeast cell wall.

[0025] The additive is used for degrading zearalenone and masked zearalenone, and the application of the additive includes removing zearalenone and masked zearalenone from feed and feed raw materials, fruit juice and pomace, grain and its processing by-products and Chinese herbal medicine in vivo and in vitro.

[0026] The beneficial effects of the present invention are as follows: the present invention provides a neutral zearalenone hydrolase HaZHD, an encoding gene, an additive and an application thereof, and particularly relates to an application thereof in degrading zearalenone and masked zearalenone; the optimal reaction pH of the zearalenone hydrolase HaZHD is 7.0; activity detection of the hydrolase HaZHD in hydrolyzing zearalenone and masked zearalenone shows that the hydrolase HaZHD can effectively degrade ZEN and masked zearalenone ZEN-14-S, ZEN-16-S, ZEN-14-G and ZEN-16-G, and has broad application prospects in production practice. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The figure is the SDS-PAGE analysis result of the purified zearalenone hydrolase HaZHD in the embodiment of the present invention;

[0028] Figure 2 HPLC analysis results of ZEN degradation by the zearalenone hydrolase HaZHD prepared in the present invention;

[0029] Figure 3 This is the degradation rate result of the hidden ZEN by the zearalenone hydrolase HaZHD prepared by the present invention;

[0030] Figure 4 The effect of pH on the activity of zearalenone hydrolase HaZHD in the enzymatic properties test of the present invention;

[0031] Figure 5 This is the effect of temperature on the activity of zearalenone hydrolase HaZHD in the enzymatic properties test of the present invention. DETAILED DESCRIPTION

[0032] The present invention will be further explained below with reference to the accompanying drawings and specific embodiments.

[0033] Example 1: Expression of Zearalenone Hydrolase HaZHD in Escherichia coli

[0034] The coding gene hazhd of the synthesized zearalenone hydrolase HaZHD was connected between the NdeI and XhoI restriction sites of the pET-31b vector, and the recombinant pET-31b-hazhd plasmid was transformed into Escherichia coli competent cells Rosetta (DE3).

[0035] The recombinant Escherichia coli Rosetta (DE3) transformed with the pET-31b-hazhd plasmid was inoculated into 5 mL of LB liquid medium and activated overnight. The culture was 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.

[0036] 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 HaZHD enzyme.

[0037] Example 2: Expression of Zearalenone Hydrolase HaZHD in Pichia pastoris

[0038] The coding gene hazhd of the synthesized zearalenone hydrolase HaZHD was ligated between the EcoRI and NotI restriction sites of the pPICZαA vector, linearized by SacI restriction enzyme, and then electroporated into Pichia pastoris competent cells X33.

[0039] The recombinant Pichia pastoris X33 transformed with the pPICZαA-hazhd plasmid was inoculated into 20 mL of BMGY liquid medium and cultured at 28°C and 200 r / min until the OD 600= 2-6, centrifuged at 4°C and 5000 rpm for 10 min, collected the bacteria and inoculated into a 500-mL Erlenmeyer flask containing 50 mL of BMMY liquid culture medium, added 0.5% methanol every 24 h to induce HaZHD expression, and cultured at 28°C and 200 rpm for 96 h. Centrifuged at 4°C and 12000 rpm for 10 min, collected the supernatant, and obtained the crude HaZHD enzyme.

[0040] Example 3: Preparation of pure zearalenone hydrolase HaZHD enzyme

[0041] The crude HaZHD enzyme solution was obtained according to the method described in Example 1, 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.

[0042] The purified protein was ultrafiltered with a 3 kDa cut-off tube to remove the imidazole contained therein, and the purified HaZHD was subjected to SDS-PAGE electrophoresis analysis. The results were as follows: Figure 1 As shown, lane 1 is the migration band of the purified HaZHD protein, with a molecular weight of approximately 30 kDa, which is consistent with the theoretical molecular weight.

[0043] Example 4: Detection of ZEN hydrolysis activity by zearalenone hydrolase HaZHD

[0044] 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 pure zearalenone hydrolase HaZHD enzyme (0.5 mg / mL) prepared in Example 3, and 10 μL ZEN stock solution. After incubation in a 37°C water bath for 0, 0.5, and 2.0 h, the reaction was terminated by adding 500 μL of methanol. The ZEN content in the reaction system was determined by HPLC.

[0045] 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.

[0046] Figure 2The figure shows the results of the activity test of zearalenone hydrolase HaZHD. It can be seen that as the reaction proceeds, the content of ZEN (corresponding to a retention time of 7.4 min) in the reaction system decreases, and a product peak is generated at a retention time of 2.1 min. The peak area increases as the reaction proceeds, indicating that the zearalenone hydrolase HaZHD has the activity of hydrolyzing ZEN.

[0047] Example 5: Detection of the activity of zearalenone hydrolase HaZHD in hydrolyzing cryptic zearalenone

[0048] Solid masked zearalenone ZEN-14-S, ZEN-16-S, ZEN-14-G, and ZEN-16-G standards were dissolved in methanol to prepare 1.5 mg / mL stock solutions. The experiment was performed using the following 500 μL reaction system: 485 μL sodium phosphate buffer (100 mM, pH 7.0), 5 μL pure zearalenone hydrolase HaZHD enzyme (0.5 mg / mL) prepared in Example 3, and 10 μL ZEN-14-S, ZEN-16-S, ZEN-14-G, or ZEN-16-G stock solution. After incubation in a 37° C. water bath for 0 and 2.0 h, 500 μL of methanol was added to terminate the reaction. The contents of ZEN-14-S, ZEN-16-S, ZEN-14-G, and ZEN-16-G in the reaction system were detected by high performance liquid chromatography analysis as described in Example 4, and the degradation rate was calculated.

[0049] like Figure 3 The results showed that the degradation rates of ZEN-14-S, ZEN-16-S, ZEN-14-G, and ZEN-16-G reached 95%, 92%, 93%, and 91%, respectively. This indicates that the zearalenone hydrolase HaZHD is capable of hydrolyzing the cryptic zearalenone ZEN-14-S, ZEN-16-S, ZEN-14-G, and ZEN-16-G.

[0050] Example 6: Enzymatic property test of zearalenone hydrolase HaZHD

[0051] To test the activity of zearalenone hydrolase (HaZHD) under different pH conditions, the following reaction system was used: 485 μL of buffer solutions at different pH values, 5 μL of pure HaZHD enzyme (0.5 mg / mL) prepared in Example 3, and 10 μL of ZEN stock solution. After incubation in a 37°C water bath for 10 min, 500 μL of methanol was added to terminate the reaction. The ZEN content in the reaction system was determined using the HPLC analysis method described in Example 4, and the relative enzyme activity was calculated. The results are shown in Figure 4. Figure 4 As shown in the figure, the optimal reaction pH of zearalenone hydrolase HaZHD is 7.0, and its relative enzyme activity is maintained above 50% in the pH range of 6.0-10.0.

[0052] To test the activity of zearalenone hydrolase HaZHD under different temperature conditions, the following reaction system was used: 485 μL sodium phosphate buffer (100 mM, pH 7.0), 5 μL pure enzyme (0.5 mg / mL) of zearalenone hydrolase HaZHD prepared in Example 3, and 10 μL ZEN stock solution; after incubation in a water bath for 10 min at different temperatures (22, 27, 32, 37, 42, 47, 52, and 57°C), 500 μL methanol was added to terminate the reaction. The ZEN content in the reaction system was detected by the HPLC analysis method in Example 4 above, and the relative enzyme activity was calculated. The results are shown in Figure 5. Figure 5 As shown in the figure, the optimum reaction temperature of zearalenone hydrolase HaZHD is 47℃, and its relative enzyme activity is maintained above 50% in the range of 27-52℃.

[0053] Example 7: An additive for degrading zearalenone and concealed zearalenone and its preparation method

[0054] A carrier accounting for 99.9% of the total mass of the additive is weighed, maltodextrin and starch in the carrier are mixed in a mass ratio of 1:2, and then zearalenone hydrolase HaZHD is mixed in a ratio of 0.1% of the total mass of the additive to obtain the additive.

[0055] Example 8: A method for removing ZEN and ZEN-14-G from corn steep liquor

[0056] The additive prepared in Example 7 was used to remove ZEN and ZEN-14-G from the corn steep liquor. 100 mL of corn steep liquor containing 5.3 μg / mL ZEN and 3.4 μg / mL ZEN-14-G was added to a 500 mL conical flask, the pH was adjusted to 7.0 with 1 M NaOH, 0.5 g of the additive described in Example 7 was added, and the mixture was reacted at 37° C. for 5 h. 10 mL of the reaction solution was purified by a solid phase extraction column, and the contents of ZEN and ZEN-14-G in the reaction system were detected by the high performance liquid chromatography method described in Example 4. The results showed that after the reaction, the contents of ZEN and ZEN-14-G in the corn steep liquor decreased to 0.04 μg / mL and 0.02 μg / mL, respectively.

[0057] The present invention provides a neutral zearalenone hydrolase HaZHD, an encoding gene, an additive, and an application thereof. The optimal reaction pH is 7.0. Activity detection of the hydrolase HaZHD in hydrolyzing zearalenone ZEN and cryptic zearalenone shows that the hydrolase HaZHD can effectively degrade ZEN and cryptic zearalenone ZEN-14-S, ZEN-16-S, ZEN-14-G, and ZEN-16-G, and has broad application prospects in production practice.

[0058] The above description is only used to illustrate the technical solution of the present invention and is not intended to limit it. Other modifications or equivalent substitutions made to the technical solution of the present invention by ordinary technicians in this field should be included in the scope of the claims of the present invention as long as they do not depart from the spirit and scope of the technical solution of the present invention.

Claims

1. Use of a neutral zearalenone hydrolase HaZHD in the hydrolysis of zearalenone and concealed zearalenone, characterized in that: The concealed zearalenone is any one or more of zearalenone-14-sulfate, zearalenone-16-sulfate, zearalenone-14-glucoside and zearalenone-16-glucoside; The amino acid sequence of the neutral zearalenone hydrolase HaZHD is shown in SEQ ID NO. 1; The nucleotide sequence of the gene encoding the neutral zearalenone hydrolase HaZHD is shown in SEQ ID NO.

2.

2. An application of an additive containing neutral zearalenone hydrolase HaZHD in the degradation of zearalenone and concealed zearalenone, characterized in that: The application of the additive includes in vitro and in vivo removal of zearalenone and hidden zearalenone in feed and feed raw materials, fruit juice and pomace, grain and its processing by-products and Chinese herbal medicine, wherein the hidden zearalenone is any one or more of zearalenone-14-sulfate, zearalenone-16-sulfate, zearalenone-14-glucoside and zearalenone-16-glucoside; The additive includes neutral zearalenone hydrolase HaZHD and a physiologically acceptable carrier thereof, wherein the amino acid sequence of the neutral zearalenone hydrolase HaZHD is shown in SEQ ID NO. 1, and the content of the neutral zearalenone hydrolase HaZHD is 0.01%-10%; the physiologically acceptable carrier includes one or more of limestone, talc, bentonite, kaolin, montmorillonite, chitosan, chitin, starch, sucrose, rice, rice bran, rice husk powder, soybean hull, wheat bran, corn cob powder, maltodextrin, cyclodextrin and yeast cell wall.

Images