Aldoketoreductase and application thereof in trichothecene toxin biotransformation process

Abstract

The invention belongs to the technical field of agricultural biology, and particularly relates to aldoketoreductase ZHAKR8 as well as a coding gene, a preparation method and application of the aldoketoreductase ZHAKR8. The amino acid sequence of the aldehyde ketoreductase ZHAKR8 provided by the invention is as shown in SEQ ID NO. 1, and the nucleotide sequence of the coding gene of the aldehyde ketoreductase ZHAKR8 is as shown in SEQ ID NO. 2. According to the aldoketoreductase provided by the invention, the 3-ketone-vomitoxin can be converted into the 3-epivomitoxin, the speed of degrading the 3-ketone-vomitoxin is high, and the toxicity of the product 3-epivomitoxin is greatly reduced. The method has huge application prospects in the fields of feed, food, Chinese herbal medicine, biomass energy production and the like.

Description

Technical Field

[0001] The present invention belongs to the field of agricultural biotechnology, and specifically relates to an aldehyde-keto reductase ZHAKR8 and its encoding gene and application, especially its application in the biotransformation process of trichothecene toxins. Background Art

[0002] Mycotoxin contamination of cereal grains worldwide is extremely serious. Mycotoxins not only cause diseases such as liver and kidney disease, enterotoxic syndrome, and reproductive failure in animals, resulting in significant economic losses to the livestock industry, but also can cause residues of toxins and their metabolites in meat, eggs, and milk, posing a serious threat to food safety and human health. Mycotoxins are a class of toxic secondary metabolites produced by the molds that contaminate cereal grains during their growth. Among these, the most serious contaminants are the trichothecenes, including deoxynivalenol (DON), 3-acetyl-deoxynivalenol (3ADON), 15-acetyl-deoxynivalenol (15ADON), T-2 toxin, nivalenol (NIV), and fusarium-X (FUS).

[0003] Research reports on enzymes that degrade trichothecenes are very scarce. Reported enzymes that oxidize DON to 3-keto-DON include the aldehyde-keto reductase from Sphingomonas sp. S3-4 and the PQQ-dependent alcohol dehydrogenase (DepA) from Devosiamutans 17-2-E-8; and the NADPH-dependent dehydrogenase (DepB) from Devosiamutans 17-2-E-8 that reduces 3-keto-DON to 3-epi-DON. Therefore, the discovery of new trichothecenes-degrading enzymes is needed for the biodegradation and detoxification of these toxins in feed and food, as well as for their detoxification during ethanol fermentation in the biomass energy industry. Summary of the Invention

[0004] The object of the present invention is to provide an aldehyde-keto reductase ZHAKR8.

[0005] Another object of the present invention is to provide a gene encoding the above-mentioned aldehyde-keto reductase ZHAKR8.

[0006] Another object of the present invention is to provide a recombinant expression vector comprising the above-mentioned aldehyde-keto reductase ZHAKR8 encoding gene.

[0007] Another object of the present invention is to provide a recombinant strain comprising the above-mentioned aldehyde-keto reductase ZHAKR8 encoding gene.

[0008] Another object of the present invention is to provide a preparation method comprising the above-mentioned aldehyde-keto reductase ZHAKR8.

[0009] Another object of the present invention is to provide the use of the above-mentioned aldehyde-keto reductase ZHAKR8 in the biotransformation process of trichothecene toxins.

[0010] Another object of the present invention is to provide a combined use of the above-mentioned aldehyde-keto reductase ZHAKR8 and alcohol dehydrogenase in the biotransformation process of trichothecene toxins.

[0011] Another object of the present invention is to provide a complex enzyme comprising the above-mentioned aldehyde-keto reductase ZHAKR8.

[0012] Another object of the present invention is to provide an application of a complex enzyme comprising the above-mentioned aldehyde-keto reductase ZHAKR8.

[0013] To achieve the above objectives, the present invention provides the following technical solutions:

[0014] One aspect of the present invention is to mine an aldehyde-ketone reductase ZHAKR8 capable of degrading 3-keto-DON from the genome of a nitrate-reducing bacterium using genome sequencing technology, and its amino acid sequence is shown in SEQ ID No.1.

[0015] The aldehyde-keto reductase ZHAKR8 encoding gene described in the present invention also falls within the protection scope of the present invention.

[0016] Another aspect of the present invention is to provide a recombinant expression vector or a recombinant host strain containing the aldehyde-keto reductase ZHAKR8 encoding gene; wherein, the recombinant expression vector can be a recombinant prokaryotic expression vector or a recombinant eukaryotic vector.

[0017] Another aspect of the present invention is to provide a method for preparing the aldehyde-keto reductase ZHAKR8,

[0018] include:

[0019] (1) The gene encoding aldehyde-keto reductase ZHAKR8 is operably linked to an expression regulatory element to construct a recombinant expression vector;

[0020] (2) The recombinant expression vector is transformed into a host strain, the host strain is cultured, the recombinant protein is induced to express, and the recombinant protein is purified.

[0021] Another aspect of the present invention is to provide a complex enzyme comprising the aldehyde-keto reductase ZHAKR8, wherein the complex enzyme further comprises an equal proportion of alcohol dehydrogenase.

[0022] The aldehyde-keto reductase ZHAKR8 and its composite enzyme provided by the present invention have very high biotransformation activity of trichothecene toxins or their metabolites, play an important role in the biotransformation process of trichothecene toxins, and have broad application prospects in the production of enzyme preparations, food, feed, Chinese herbal medicine or biomass energy.

[0023] Definitions of terms used in this invention

[0024] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0025] The term "host cell" or "recombinant host cell" means a cell comprising a polynucleotide of the present invention, regardless of the method used for insertion to produce the recombinant host cell, such as direct uptake, transduction, f-mating, or other methods known in the art. The exogenous polynucleotide may be maintained as a non-integrating vector, such as a plasmid, or may be integrated into the host genome.

[0026] The term "transformation" refers to the process by which a eukaryotic cell acquires new genetic markers as a result of the incorporation of foreign DNA. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] 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 the embodiments or the description of the prior art:

[0028] Figure 1 Shown is the SDS-PAGE electrophoresis analysis of the expression product of the recombinant plasmid pET-31b-ZHAKR8 after purification;

[0029] Figure 2 The figure shows the degradation of 3-keto-vomitoxin by ZHAKR8 protein under different pH conditions.

[0030] Figure 3 Shown is the degradation of 3-keto-vomitoxin by ZHAKR8 protein under different temperature conditions;

[0031] Figure 4 Shown is the stability effect diagram of ZHAKR8 protein at different pH;

[0032] Figure 5 Shown is the stability effect of ZHAKR8 protein at different temperatures. DETAILED DESCRIPTION

[0033] The present invention will be further described below with reference to specific embodiments. However, these embodiments are merely exemplary and do not limit the scope of the present invention in any way. It should be understood by those skilled in the art that the details and forms of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, and such modifications and replacements fall within the scope of protection of the present invention.

[0034] Example 1 Acquisition and expression of aldehyde-keto reductase ZHAKR8 protein

[0035] The genomic DNA of nitrate-reducing Pelagobacterium ZH15 was used as an amplification template to amplify the gene encoding the ZHAKR8 protein. A recombinant expression vector containing the ZHAKR8 protein encoding gene sequence and its engineered bacteria were constructed to express the ZHAKR8 protein.

[0036] The specific steps are as follows:

[0037] 1. Cloning of the ZHAKR8 gene encoding aldehyde-keto reductase

[0038] First, genomic DNA from the nitrate-reducing Bacillus pelagicus was extracted. The gene encoding the aldehyde-keto reductase ZHAKR8 was then amplified: Based on the multiple cloning site of the pET-31b vector, NdeⅠ and XhoⅠ were selected as restriction sites. Upstream primer P1 and downstream primer P2 were designed and synthesized by Shanghai Sangon Biotechnology Co., Ltd. The sequences of upstream primer P1 and downstream primer P2 were designed as follows:

[0039] Upstream primer P1: 5′-GGAGATATACATATGAGCGAAGTCAACGCC-3′

[0040] Downstream primer P2: 5′-GTGGTGGTGGTGCTCGAGTTTGCGCCCGG-3′

[0041] PCR amplification was performed using the genomic DNA of the nitrate-reducing bacterium Pelagobacterium as a template. The reaction conditions are shown in Table 1:

[0042] Table 1 PCR amplification reaction conditions

[0043] DNA template 1 μL Upstream primer P1 2μL Downstream primer P2 2μL 2×PfuPCRMix 25 μL <![CDATA[ddH2O]]> 20 μL Total volume 50 μL

[0044] Amplification conditions were as follows: 95°C pre-denaturation for 5 minutes, 95°C denaturation for 30 seconds, 56°C annealing for 30 seconds, 72°C extension for 2 minutes for 30 cycles, and 72°C full extension for 10 minutes. PCR products were electrophoresed on a 1% agarose gel and recovered using an agarose gel DNA recovery kit.

[0045] 2. Construction of a recombinant expression vector containing the gene sequence encoding aldehyde-keto reductase ZHAKR8

[0046] Preparation of linearized vector: Double digest the pET-31b plasmid with NdeⅠ and XhoⅠ. The enzyme digestion system is shown in Table 2:

[0047] Table 2 Enzyme digestion system

[0048] pET-31b plasmid 30 μL NdeⅠ 1 μL XhoⅠ 1 μL 10×CutSmartBuffer 5μL <![CDATA[ddH2O]]> 13μL Total volume 50 μL

[0049] Enzyme digestion conditions: 37°C water bath for 30 min. The digestion products were electrophoresed on 1% agarose gel, and the digested vector was recovered using an agarose gel DNA recovery kit.

[0050] Homologous recombination cloning: The recombinant expression vector was constructed using the Adlay one-step seamless cloning kit. The reaction system is shown in Table 3:

[0051] Table 3 Reaction system constructed by recombinant expression vector

[0052] 2×OneStepCloningMix 5μL Linearized pET-31b plasmid 2μL PCR products 2μL <![CDATA[ddH2O]]> 1 μL Total volume 10 μL

[0053] Reaction conditions: Mix gently and react at 50°C for 30 minutes. After the reaction, place the PCR tube on ice and transform the ligation product into competent E. coli DH5α cells. Screen for ampicillin resistance, select positive transformants, extract the recombinant plasmid, and perform single and double enzyme digestion verification and sequencing to confirm the correct recombinant strain. Then, transform the correct recombinant plasmid into E. coli TSSeta(DE3)pLysS.

[0054] 3. Inducible expression and purification of aldehyde-keto reductase ZHAKR8 in Escherichia coli

[0055] The recombinant Escherichia coli TSSeta (DE3) pLysS transformed with the pET-31b-ZHAKR8 plasmid was inoculated into 5 mL of liquid LB medium for activation overnight, and then transferred to a 500 mL Erlenmeyer flask with a liquid volume of 1 L at a ratio of 1:100. The culture was cultured at 180 r / min and 37°C until the OD600 reached 0.7. A final concentration of 0.4 mM IPTG was added to induce protein expression.

[0056] The fermentation broth was collected, centrifuged at 4°C, 12000r / min for 30min, and the supernatant was discarded; the bacteria were resuspended with PBS solution of pH 7.4, centrifuged at 4°C, 12000r / min for 30min, the supernatant was discarded, and the washing of the bacteria was repeated three times. The bacterial cells were then resuspended in binding buffer, ultrasonically disrupted, centrifuged at 4°C, 12000r / min for 10min, the supernatant was collected and filtered. Since the expressed ZHAKR8 protein has 6 His tags at the C-terminus, a nickel ion affinity chromatography column was used to purify the protein. For the steps of equilibration, loading, elution, etc., please refer to the Qiagen user manual. The purified protein was ultrafiltered with a cutoff tube (10kDa) to remove the imidazole contained therein, and the target band was detected by SDS-PAGE. The results are shown as follows. Figure 1 As shown, lane 5 is the expression product, and the arrow indicates the target band, which indicates that the molecular weight of the protein expressed by the recombinant strain is about 31 kDa, which is consistent with the theoretical molecular weight.

[0057] Example 2 Effect of pH on the activity of aldehyde-keto reductase ZHAKR8 in degrading 3-keto-donitrotoxin

[0058] Solid 3-keto-DON was dissolved in acetonitrile to prepare a 1 mg / mL stock solution, and the prosthetic group NADPH was dissolved in ultrapure water to prepare a 10 mM stock solution. To test the effect of the aldehyde-keto reductase ZHAKR8 on the degradation of 3-keto-DON under different pH conditions, the following 500 μL reaction system was used: 455 μL of different pH buffers (100 mM sodium citrate buffer, pH 4-6; 100 mM sodium phosphate buffer, pH 7-8; 100 mM glycine-NaOH buffer, pH 9-10), 5 μL of purified ZHAKR8 protein (10.65 μg), 25 μL of the prosthetic group NADPH solution, and 15 μL of the 3-keto-DON solution. The reaction was carried out at 37°C for 150 min, and then 500 μL of methanol was added to terminate the reaction. The mixture was centrifuged at 12,000 rpm for 1 min, and the supernatant was filtered using a Millex-GV filter membrane (0.22 μm). The residual 3-keto-domitoxin content in the system was detected by high performance liquid chromatography.

[0059] The chromatographic conditions for the high performance liquid chromatography detection of 3-keto-vomitoxin were as follows: chromatographic column: Agilent C18 column, 4.6 mm × 150 mm × 5 μm; acetonitrile-water (23:77); flow rate: 1 mL / min; pump pressure: 100 bar; injection volume: 20 μL; UV detector detection wavelength: λ = 218 nm; acquisition time: 10 min.

[0060] Degradation rate of 3-keto-DON (%) = (1-remaining amount of 3-keto-DON in the treatment group / amount of 3-keto-DON in the control group) × 100%

[0061] The degradation rate of 3-keto-DON at the optimal pH was taken as 100%, and the relative activity of aldehyde-keto reductase ZHAKR8 in degrading 3-keto-DON under other conditions was calculated.

[0062] The results are as follows Figure 2 As shown, the optimal pH for the degradation of 3-keto-DON by aldehyde-keto reductase ZHAKR8 is 9.0.

[0063] Example 3 Effect of Temperature on the Degradation Activity of 3-Keto-DON by Aldo-Keto Reductase ZHAKR8

[0064] The effect of different temperatures on the degradation of 3-keto-DON by the aldehyde-keto reductase ZHAKR8 was determined in a 500 μL reaction system containing 455 μL of sodium phosphate buffer (100 mM, pH 7), 5 μL of purified ZHAKR8 protein (10.65 μg), 25 μL of NADPH solution, and 15 μL of 3-keto-DON solution. A control without ZHAKR8 protein was used. The reaction was carried out at various temperatures (20, 30, 40, 50, 60, 70, and 80°C) for 150 min. The reaction was terminated by the addition of 500 μL of methanol, followed by centrifugation at 12,000 rpm for 1 min. The supernatant was filtered through a Millex-GV filter (0.22 μm) and the residual 3-keto-DON content was determined by high-performance liquid chromatography. The degradation rate of 3-keto-DON at the optimal temperature was 100%, and the relative activity of aldehyde-keto reductase ZHAKR8 in degrading 3-keto-DON under other conditions was calculated.

[0065] The results are as follows Figure 3 As shown, the optimal temperature for the degradation of 3-keto-DON by aldehyde-keto reductase ZHAKR8 is 50℃.

[0066] Example 4 pH stability of aldehyde-keto reductase ZHAKR8

[0067] To test the pH stability of the ZHAKR8 aldehyde-keto reductase, the residual activity of the ZHAKR8 recombinant protein in degrading 3-keto-DON was determined after treatment in different pH buffers for varying durations. Seven pH levels (3, 4, 5, 6, 7, 8, and 9) were set, with two treatment durations (12 h and 24 h). After treatment at different pH conditions for varying durations, the residual activity was determined under optimal conditions. The reaction system (500 μL) consisted of 455 μL of pH 7 buffer (100 mM sodium phosphate buffer), 5 μL of ZHAKR8 aldehyde-keto reductase (10.65 μg), 25 μL of NADPH, and 15 μL of 3-keto-DON solution (final concentration 30 ppm). The reaction was incubated at 37°C for 150 min, then terminated with 500 μL of HPLC-grade methanol. The 3-keto-DON content in the samples was determined by high-performance liquid chromatography (HPLC), and the degradation rate was calculated. The degradation rate at 0 min was defined as 100%, and the relative activity under different conditions was calculated.

[0068] The results are as follows Figure 4 As shown, the aldehyde-keto reductase ZHAKR8 has good pH stability in a neutral environment, and the enzyme still has 71.1% residual activity after being treated in pH = 7 buffer for 24 hours.

[0069] Example 5 Temperature stability of aldehyde-keto reductase ZHAKR8

[0070] To test the temperature stability of the ZHAKR8 aldehyde-keto reductase, the residual activity of the recombinant ZHAKR8 protein in degrading 3-keto-DON was determined after treatment at different temperatures and for different times. Six temperature levels (25, 30, 35, 40, 45, and 50°C) were set, with treatment times of 0.5, 1, 1.5, and 2 h. After treatment at different temperatures and for different times, the sample was placed on ice for 1 min, and the residual activity was determined under the optimal conditions. The reaction system (500 μL) consisted of 455 μL of pH 7 buffer (100 mM sodium phosphate buffer), 5 μL of ZHAKR8 aldehyde-keto reductase (10.65 μg), 25 μL of NADPH, and 15 μL of 3-keto-DON solution (final concentration 30 ppm). The reaction was incubated at 37°C for 150 min, then terminated by the addition of 500 μL of HPLC-grade methanol. The 3-keto-DON content in the sample was determined by high-performance liquid chromatography (HPLC), and the degradation rate was calculated. The degradation rate at 0 min of treatment was defined as 100%, and the relative activities under different conditions were calculated.

[0071] The results are as follows Figure 5 As shown, the aldehyde-keto reductase ZHAKR8 has good stability below 45℃, with 83.4% relative activity remaining after treatment at 35℃ for 2h, 64.5% relative activity remaining after treatment at 45℃ for 2h, and 27.2% relative activity remaining after treatment at 50℃ for 2h.

[0072] Example 6 Aldo-keto reductase ZHAKR8 combined with alcohol dehydrogenase ZHDDH for biotransformation of vomitoxin

[0073] To test the combined activity of ZHAKR8 and ZHAKR8 for the biotransformation of DON, a 500 μL reaction system was prepared: 170 μL of pH 7.0 buffer (100 mM sodium phosphate buffer), 270 μL of ZHAKR8 (100 μg), 25 μL of PQQ, 5 μL of ZHAKR8 (10.65 μg), 25 μL of NADPH, and 5 μL of DON stock solution (final concentration 50 μg / mL). A control without ZHAKR8 and ZHAKR8 was used. The reaction was incubated at 37°C for 48 h and terminated with 500 μL of HPLC-grade methanol. The DON biomass content in the sample was determined by high-performance liquid chromatography (HPLC), and the degradation rate was calculated.

[0074] The results showed that the efficiency of aldehyde-keto reductase ZHAKR8 combined with alcohol dehydrogenase ZHDDH in catalyzing the degradation of vomitoxin was 95.4%.

[0075] Example 7 Aldo-keto reductase ZHAKR8 combined with alcohol dehydrogenase ZHDDH for biotransformation of 15-acetyldeoxynivalenol

[0076] To test the biotransformation of 15-acetyldeoxynivalenol by combining the aldehyde-keto reductase ZHAKR8 with the alcohol dehydrogenase ZHDDH, a 500 μL reaction system was used: 170 μL of pH 7.0 buffer (100 mM sodium phosphate buffer), 270 μL of alcohol dehydrogenase ZHDDH (100 μg), 25 μL of PQQ, 5 μL of aldehyde-keto reductase ZHAKR8 (10.65 μg), 25 μL of NADPH, and 5 μL of a 15-acetyldeoxynivalenol stock solution (final concentration 10 μg / mL). A control without ZHDDH and ZHAKR8 proteins was used. The reaction was incubated at 37°C for 48 h and terminated with 500 μL of HPLC-grade methanol. The biomass of DON in the sample was determined by high-performance liquid chromatography (HPLC), and the degradation rate was calculated.

[0077] The results showed that the efficiency of aldehyde-keto reductase ZHAKR8 combined with alcohol dehydrogenase ZHDDH in catalyzing the degradation of 15-acetyldeoxynivalenol was 98.2%.

[0078] Example 8 Aldo-keto reductase ZHAKR8 combined with alcohol dehydrogenase ZHDDH for biotransformation of nivalenol

[0079] To test the biotransformation of nivalenol by combining the aldehyde-keto reductase ZHAKR8 with the alcohol dehydrogenase ZHDDH, a 500 μL reaction system was prepared: 170 μL pH 7.0 buffer (100 mM sodium phosphate buffer), 270 μL alcohol dehydrogenase ZHDDH (100 μg), 25 μL PQQ, 5 μL aldehyde-keto reductase ZHAKR8 (10.65 μg), 25 μL NADPH, and 5 μL nivalenol stock solution (final concentration 10 μg / mL). A control without ZHDDH and ZHAKR8 proteins was used. The reaction was incubated at 37°C for 48 h and terminated with 500 μL HPLC-grade methanol. The nivalenol biomass in the sample was determined by high-performance liquid chromatography (HPLC), and the degradation rate was calculated.

[0080] The results showed that the efficiency of aldehyde-keto reductase ZHAKR8 combined with alcohol dehydrogenase ZHDDH in catalyzing the degradation of nivalenol was 46.3%.

[0081] Example 9 Preparation of a complex enzyme

[0082] Aldo-keto reductase ZHAKR8 and alcohol dehydrogenase ZHDDH are mixed in equal proportions and then mixed with an appropriate amount of a carrier to form a complex enzyme. The carrier can be one or more of maltodextrin, cyclodextrin, sucrose, starch, wheat, wheat bran, rice, rice bran, corn gluten meal, montmorillonite, zeolite powder, or yeast cell walls. The enzyme-to-carrier ratio can be 1:1-10.

[0083] Example 10: Effect of complex enzyme on detoxification of trichothecenes in feed

[0084] The feed containing trichothecenes treated with the composite enzyme described in Example 9 (5 ppm of vomitoxin, 1 ppm of 15-acetyldeoxynivalenol, and 1 ppm of nivalenol) was mixed at a ratio of 0.1% and digested in simulated animal gastrointestinal fluid in vitro for 24 hours to degrade the trichothecenes in the feed.

[0085] Simulated gastric fluid: Accurately weigh 2 g of feed containing vomitoxin, 15-acetyldeoxynivalenol, and nivalenol, add 2 mg of the trichothecene-degrading enzyme ZHDDH4 or its single-site mutant N95M, place in a 100 mL Erlenmeyer flask, add 25 mL of 0.1 M PBS (pH 6.0), adjust the pH to 6.8, and mix thoroughly. Add 1 mL of prepared amylase solution and digest at 39°C, 150 rpm, for 2 h. Add 10 mL of 0.2 M HCl, adjust the pH to 2.0 with 1 M HCl or 1 M NaOH solution, add 1 mL of freshly prepared acid protease (50,000 U / g), mix thoroughly, seal with parafilm, and incubate in a shaker at 39°C, 150 rpm, for 6 h.

[0086] Simulated small intestinal fluid: After incubation with simulated gastric fluid for 6 h, add 5 mL of 0.6 M NaOH solution, adjust the pH to 6.8 with 1 M HCl or 1 M NaOH solution, add freshly prepared intestinal exogenous enzyme suspension (protease: amylase: lipase = 3:1:1), seal with parafilm, and incubate in a 39°C constant temperature shaker for 18 h (150 rpm).

[0087] After the reaction, the degradation rate of the complex enzyme on trichothecenes was measured. The results showed that the degradation rate of vomitoxin was 82.4%, the degradation rate of 15-acetyldeoxynivalenol was 90.2%, and the degradation rate of nivalenol was 34.5%.

[0088] Example 11: Detoxification effect of complex enzymes on trichothecenes in DDGS, a byproduct of ethanol fermentation

[0089] The complex enzyme-treated DDGS containing trichothecenes (7 ppm of vomitoxin, 2 ppm of 15-acetyldeoxynivalenol, and 1.5 ppm of nivalenol) described in Example 9 was mixed at a ratio of 0.1% and digested in simulated animal gastrointestinal fluid in vitro for 24 h to degrade the trichothecenes in DDGS.

[0090] After the reaction, the degradation rate of the complex enzyme on trichothecenes was measured. The results showed that the degradation rate of vomitoxin was 78.3%, the degradation rate of 15-acetyldeoxynivalenol was 87.5%, and the degradation rate of nivalenol was 30.2%.

[0091] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. An aldehyde-keto reductase ZHAKR8 with biological detoxification function, characterized in that: Its amino acid sequence is as shown in SEQ ID NO.

1.

2. The gene encoding the aldehyde-keto reductase ZHAKR8 according to claim 1, characterized in that Its nucleotide sequence is as shown in SEQ ID NO.

2.

3. A recombinant expression vector or recombinant strain comprising the aldehyde-keto reductase ZHAKR8 gene according to claim 2, The recombinant strain is characterized in that The recombinant strain includes Escherichia coli, yeast, Aspergillus, Bacillus or Lactobacillus.

4. The method for preparing the aldehyde-keto reductase ZHAKR8 according to claim 1, characterized in that: The method comprises the following steps: (1) transforming a host cell with a recombinant expression vector encoding an aldehyde-keto reductase ZHAKR8 gene to obtain a recombinant strain; (2) Cultivating the recombinant strain and inducing the expression of the aldehyde-keto reductase ZHAKR8 protein; (3) Isolation and purification of aldehyde-keto reductase ZHAKR8.

5. The use of the aldehyde-keto reductase ZHAKR8 in the biotransformation process of trichothecene toxins according to claim 1, characterized in that: The trichothecene toxins are one or more of vomitoxin, 15-acetyldeoxynivalenol, nivalenol, and their metabolites 3-keto-vomitoxin, 3-keto-15-acetyldeoxynivalenol, and 3-keto-nivalenol.

6. The combined use of aldehyde-keto reductase ZHAKR8 and alcohol dehydrogenase in the biotransformation process of trichothecene toxins, characterized in that: The aldehyde-keto reductase ZHAKR8 is the enzyme according to claim 1; The alcohol dehydrogenase is an enzyme that can dehydrogenate trichothecene toxins to produce ketotrichothecene toxins; The trichothecene toxins are one or more of vomitoxin, 15-acetyldeoxynivalenol, nivalenol, and their metabolites 3-keto-vomitoxin, 3-keto-15-acetyldeoxynivalenol, and 3-keto-nivalenol.

7. A composite enzyme, prepared by compounding the aldehyde-keto reductase ZHAKR8 according to claim 1 and alcohol dehydrogenase in equal proportions.

8. Use of the aldehyde-keto reductase ZHAKR8 according to claim 1 or the complex enzyme according to claim 7 in the production of food, feed, Chinese herbal medicine or biomass energy.

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