Aflatoxin B1 degrading enzyme as well as coding gene and application thereof

Abstract

The invention belongs to the technical field of agricultural biology, and particularly relates to an aflatoxin B1 degrading enzyme as well as a coding gene and application thereof. The amino acid sequences of the degrading enzymes PcAFB1A and GmAFB1A are respectively shown as SEQ ID NO.1 and SEQ ID NO.2, and the coding gene sequences of the degrading enzymes PcAFB1A and GmAFB1A are respectively shown as SEQ ID NO.3 and SEQ ID NO.4; the two enzymes can degrade aflatoxin B1 (AFB1) under mild conditions, the degradation rates are 36.58% and 34.93% respectively, and the degradation rate reaches 41.18% when the two enzymes are used in a composite mode. The strain also has a certain detoxification capability on a complex material, namely mildewed corn, polluted by high-concentration AFB1, and the degradation rate on the AFB1 in the material reaches 25.17%; according to the invention, an AFB1 biodegradation enzyme library is enriched, development of an AFB1 efficient degradation compound enzyme is promoted, and the method has potential application value in detoxification of AFB1 polluted food and feed.

Description

Technical Field

[0001] The invention belongs to the field of agricultural biotechnology, and particularly relates to an aflatoxin B1 degrading enzyme. Background Art

[0002] Aflatoxin B1 (AFB1) is a highly toxic secondary metabolite produced by fungi such as Aspergillus flavus and Aspergillus parasiticus. It is highly carcinogenic, teratogenic, and mutagenic. Among cereals, corn, due to its high starch content and suitable storage conditions, is a major source of AFB1 contamination. According to statistics, approximately 25% of global cereal production is contaminated with AFB1 annually. Direct use of contaminated corn as feed not only hinders the growth and development of livestock and poultry and weakens their immunity, but also accumulates through the food chain, posing a threat to human health and causing significant economic losses.

[0003] Currently, treatment technologies for AFB1 contamination primarily include physical adsorption, chemical degradation, and biological treatment. Physical adsorption often utilizes adsorbents such as montmorillonite and activated carbon, but this method only adsorbs and fixes the toxin, failing to fundamentally eliminate the toxicity of AFB1 and potentially adsorbing nutrients from feed. Chemical degradation, while capable of destroying the structure of AFB1, uses chemical reagents that can introduce secondary contamination, impacting the quality and safety of agricultural products. While microbial fermentation offers environmental advantages, it suffers from long strain screening cycles and inconsistent degradation efficiency, making it difficult to meet practical production needs. Enzymatic methods, in which degradative enzymes convert toxins into low- or non-toxic products through enzymatic reactions, offer significant advantages over other degradation methods in terms of specificity, efficiency, and environmental friendliness, making them a research hotspot for AFB1 pollution prevention and control. However, currently available AFB1 detoxification enzymes remain scarce, and existing research on AFB1-degrading enzymes has primarily focused on a few known microorganisms, such as Bacillus and white-rot fungi. Therefore, continued development of AFB1-degrading enzymes is urgently needed. Summary of the Invention

[0004] In order to promote the biodegradation of AFB1, the present invention provides an aflatoxin B1 degrading enzyme, its encoding gene and application.

[0005] The technical solution of the present invention is achieved as follows: On the one hand, the present invention applies to protect an aflatoxin B1 degrading enzyme, which is a degrading enzyme Pc AFB1A or degradation enzyme Gm AFB1A, a degrading enzyme Pc The amino acid sequence of AFB1A is shown in SEQ ID NO.1. Gm The amino acid sequence of AFB1A is shown in SEQ ID NO.2.

[0006] Wild-type aflatoxin B1 degrading enzyme of the present invention Pc AFB1A, whose amino acid sequence is shown in SEQ ID No. 1: This enzyme gene encodes 696 amino acids and has no signal peptide sequence; mature aflatoxin B1 degrading enzyme Pc The theoretical molecular weight of AFB1A is 77.85 kDa.

[0007] Wild-type aflatoxin B1 degrading enzyme of the present invention Gm AFB1A, whose amino acid sequence is shown in SEQ ID No. 2; the enzyme gene encodes 658 amino acids and has no signal peptide sequence; mature aflatoxin B1 degrading enzyme Gm The theoretical molecular weight of AFB1A is 72.94 kDa.

[0008] In the second aspect, the present invention applies to protect the above-mentioned aflatoxin B1 degrading enzyme encoding gene, the degrading enzyme Pc The nucleotide sequence of the AFB1A coding gene is shown in SEQ ID NO.3, and the degradation enzyme Gm The nucleotide sequence of the gene encoding AFB1A is shown in SEQ ID NO.4.

[0009] The target gene of the present invention is derived from the currently published Phanerochaetecarnosa and Galena marginale Galerinamarginata The genome sequence of the fungus was used as a control; the fungal genome mining technology was used to mine potential AFB1 degrading enzyme genes from the genome of the above fungi, and the mined genes encoding potential aflatoxin B1 degrading enzymes were manually inspected and corrected. Pc AFB1A and Gm Genetic model of AFB1A; Aflatoxin B1 degrading enzyme Pc The full length of the AFB1A gene sequence is 2088 bp; aflatoxin B1 degrading enzyme Gm The full length of the AFB1A encoding gene sequence is 1974 bp.

[0010] In the third aspect, the present invention applies to protect a recombinant vector comprising the above-mentioned encoding gene, wherein the above-mentioned recombinant vector is pPICZαA- Pc AFB1A or pPICZαA- Gm AFB1A.

[0011] The aflatoxin B1 degrading enzyme gene fragment synthesized by the present invention is inserted into the appropriate restriction enzyme cutting sites of the expression vector so that the nucleotide sequence thereof can be operably connected with the expression regulatory sequence; the aflatoxin B1 degrading enzyme gene fragment of the present invention is inserted into the appropriate restriction enzyme cutting sites of the expression vector so that the nucleotide sequence thereof can be operably connected with the expression regulatory sequence; Pc AFB1A and Gm AFB1A was inserted between the NotI / EcoRI restriction enzyme sites on the plasmid pPICZαA, so that the nucleotide sequence was located downstream of the AOX promoter and was regulated by it. The Escherichia coli DH5α cloning host was transformed, the plasmid was extracted, and sequencing was performed to obtain the recombinant expression plasmid pPICZαA- Pc AFB1A and pPICZαA- Gm AFB1A.

[0012] In the fourth aspect, the present invention applies to protect a recombinant strain comprising the above-mentioned encoding gene, wherein the above-mentioned recombinant strain is Pichia pastoris X33 / Pc AFB1A or Pichia pastoris X33 / Gm AFB1A.

[0013] In a fifth aspect, the present invention applies to protect a method for preparing an aflatoxin B1 degrading enzyme, comprising the following steps: (1) Transforming the above-mentioned recombinant vector into a host cell to obtain a recombinant strain; (2) The seed liquid of the recombinant strain was inoculated into BMGY medium and cultured for 24 h. The culture was then centrifuged and the bacterial pellet was resuspended in BMMY induction medium and supplemented with methanol for 3 days. (3) After the culture is completed, centrifugation is performed and the resulting supernatant is aflatoxin B1 degrading enzyme; The above recombinant vector is pPICZαA- Pc When AFB1A is expressed, the supernatant is the degrading enzyme Pc AFB1A; the recombinant vector is pPICZαA- Gm When AFB1A is expressed, the supernatant is the degrading enzyme Gm AFB1A.

[0014] Preferably, the above steps are as follows: the obtained aflatoxin B1 degrading enzyme Pc AFB1A and Gm The recombinant expression plasmid pPICZαA- Pc AFB1A and pPICZαA- Gm AFB1A was transformed into Pichia pastoris X-33 to obtain recombinant Pichia pastoris engineering bacteria X33 / Pc AFB1A and X33 / Gm AFB1A; Take X33 / Pc AFB1A and X33 / Gm The AFB1A strain was inoculated into 50 mL of YPD culture medium and cultured overnight at 30°C and 180 rpm. Subsequently, the culture medium was inoculated into 300 mL of BMGY liquid medium at a 2% inoculum volume and cultured in a shaking incubator at 30°C and 180 rpm for 24 h until the OD value of the bacterial solution reached 0. 600 The value reached 3-6; finally, the bacteria were collected by centrifugation (5000 rpm, 4°C, 10 min), the supernatant was discarded, and the bacteria were resuspended in 200 mL BMMY induction medium. The culture was continued at 22°C and 200 rpm, and 1% methanol was added every 24 h. The culture was continued for 3 days. Finally, the bacteria were centrifuged (5000 rpm, 4°C, 10 min) to remove the bacterial sediment and the supernatant was collected. Pc AFB1A or Gm AFB1A crude protein.

[0015] In a sixth aspect, the present invention applies to protect a detoxification enzyme preparation, including any of the following situations: a. Degradation enzymes Pc AFB1A is the active ingredient; b. Degradation enzymes Gm AFB1A is the active ingredient; c. Degradation enzymes Pc AFB1A and degrading enzymes Gm AFB1A is the active ingredient.

[0016] Preferably, the above-mentioned degradative enzyme Pc The amino acid sequence of AFB1A is shown in SEQ ID NO.1. Gm The amino acid sequence of AFB1A is shown in SEQ ID NO. 2; Preferably, the degradation enzyme in the above situation a Pc The concentration of AFB1A was 2.48 mg / mL, and the degradation enzyme in case b Gm The concentration of AFB1A was 2.12 mg / mL; in case c, the degradation enzyme Pc AFB1A and degrading enzymes Gm The volume ratio of AFB1A when used in combination was 1:1, and the concentrations were 2.48 mg / mL and 2.12 mg / mL, respectively.

[0017] In a seventh aspect, the present invention applies to protect the use of the above-mentioned aflatoxin B1 degrading enzyme, the above-mentioned encoding gene, the above-mentioned recombinant vector, the above-mentioned recombinant strain, or the above-mentioned detoxification enzyme preparation in mycotoxin detoxification.

[0018] Preferably, the mycotoxin is aflatoxin B1 in moldy corn, a complex material contaminated with high concentrations of AFB1.

[0019] Preferably, the above detoxification conditions are mild, and the optimal conditions are reaction at 35° C. and pH=6.0 for 36 h.

[0020] The present invention has the following beneficial effects: Aflatoxin B1 degrading enzyme provided by the present invention Pc AFB1A and Gm AFB1A enriches the currently available AFB1 enzyme detoxification resource library, which can degrade AFB1 under mild conditions, with an optimal temperature of 35°C, an optimal pH of 6.0, and an optimal reaction time of 36 h. Pc AFB1A and Gm The degradation rates of AFB1A for AFB1 were 36.58% and 34.93%, respectively, and the degradation rate of AFB1 reached 41.18% when used synergistically in equal volumes; the removal efficiency of AFB1 in complex materials - moldy corn was 19.43% and 18.65%, respectively, and the degradation rate of AFB1 in moldy corn reached 25.17% when used synergistically in equal volumes; this application provides more options for the biodegradation of AFB1, promotes the development of efficient AFB1 degradation complex enzymes, and shows potential application prospects in detoxifying AFB1-contaminated food and feed. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] 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.

[0022] 图1 Recombinant aflatoxin B1 degrading enzyme Pc AFB1A and Gm The optimal reaction temperature for AFB1A degradation of AFB1.

[0023] 图2 Recombinant aflatoxin B1 degrading enzyme Pc AFB1A and Gm Optimal reaction pH of AFB1A.

[0024] 图3 Recombinant aflatoxin B1 degrading enzyme Pc AFB1A and Gm Optimal response time of AFB1A.

[0025] 图4 Recombinant aflatoxin B1 degrading enzyme Pc AFB1A and Gm AFB1A removal efficiency of AFB1.

[0026] 图5 Recombinant aflatoxin B1 degrading enzyme Pc AFB1A and Gm AFB1A removal efficiency of AFB1 in moldy corn. DETAILED DESCRIPTION

[0027] The technical solutions of the present application will be clearly and completely described below in combination with the embodiments of the present application. Obviously, the described embodiments are only some of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative effort belong to the scope of protection of the present application.

[0028] The experimental methods used in the following experimental examples are conventional methods unless otherwise specified; the materials, reagents, etc. used are reagents and materials available through commercial channels unless otherwise specified.

[0029] With the rapid development of genome sequencing technology, a large number of fungal genome sequences have been published in the JGI Mycocosm database, which provides the possibility of mining potential new fungal degrading enzymes through bioinformatics means. The present application makes full use of other potential toxin-degrading microbial resources in the strain library, and targets to obtain AFB1 degrading enzyme coding genes from the published genomes, so as to identify new AFB1 degrading enzymes. The invention will enrich the current available AFB1 biological enzyme detoxification resource library, and promote the creation and application of AFB1 high-efficiency degrading enzyme preparations.

[0030] Experimental materials and reagents: (1) Genes and vectors: Pichia pastoris expression vector pPICZαA and strain X-33; (2) Enzymes and other biochemical reagents: endonuclease, recombinase, aflatoxin B1, moldy corn; (3) Culture medium: Yeast activation YPD culture medium: yeast extract powder 3.6 g, peptone 7.2 g, glucose 7.2 g, water 360 mL, 1 M potassium phosphate buffer pH 6 30 mL, 10×amino-free yeast nitrogen source 30 mL, biotin 0.6 mL; Yeast culture BMGY medium: yeast extract powder 3 g, peptone 6 g, 10% glycerol 30 mL, water 210 mL; Yeast induction BMMY medium: yeast extract powder 2.4 g, peptone 4.8 g, water 192 mL, 1 M potassium phosphate buffer pH 6, 10×amino-free yeast nitrogen source, 0.96 mL biotin, 1.44 mL methanol.

[0031] Example 1: Aflatoxin B1 degrading enzyme gene recombinant expression vector pPICZαA- Pc AFB1A and pPICZαA- Gm Construction of AFB1A The target gene of the present invention is derived from the currently published Phanerochaetecarnosa and Galena marginale Galerinamarginata Using currently available fungal AFB1-degrading enzymes as controls, fungal genome mining technology was used to mine potential AFB1-degrading enzyme genes from the genomes of the above fungi.

[0032] Manual inspection and correction of the mined aflatoxin B1-degrading enzymes Pc AFB1A and Gm The gene model of AFB1A was prepared, and NotI / EcoRI restriction sites were added at both ends of the gene, which was then sent to Universal Biotechnology Co., Ltd. for synthesis.

[0033] Subsequently, the synthesized aflatoxin B1 degrading enzyme gene fragment (SEQ ID NO.3 or SEQ ID NO.4) was connected to the NotI / EcoRI double-digested pPICZαA vector by homologous recombination, transformed into Escherichia coli DH5α cloning host, and the plasmid was extracted and sequenced to obtain the aflatoxin B1 degrading enzyme gene recombinant expression vector pPICZαA- Pc AFB1A and pPICZαA- Gm AFB1A.

[0034] Example 2: Aflatoxin B1 degrading enzyme Pc AFB1A and Gm Preparation of AFB1A The obtained aflatoxin B1 degrading enzyme Pc AFB1A and Gm The recombinant expression plasmid pPICZαA- Pc AFB1A and pPICZαA- Gm AFB1A was transformed into Pichia pastoris X-33 to obtain recombinant Pichia pastoris engineering bacteria X33 / Pc AFB1A and X33 / Gm AFB1A.

[0035] Take X33 / Pc AFB1A and X33 / Gm The AFB1A strain was inoculated into 50 mL of YPD culture medium and cultured overnight at 30°C and 180 rpm. Subsequently, the culture medium was inoculated into 300 mL of BMGY liquid medium at a 2% inoculum volume and cultured in a shaking incubator at 30°C and 180 rpm for 24 h until the OD value of the bacterial solution reached 0. 600 The value reached 3-6; finally, the bacteria were collected by centrifugation (5000 rpm, 4°C, 10 min), the supernatant was discarded, and the bacteria were resuspended in 200 mL BMMY induction medium. The culture was continued at 22°C and 200 rpm, and 1% methanol was added every 24 h. The culture was continued for 3 days. Finally, the bacteria were centrifuged (5000 rpm, 4°C, 10 min) to remove the bacterial sediment and the supernatant was collected. Pc AFB1A or Gm AFB1A crude protein.

[0036] Example 3: Recombinant aflatoxin B1 degrading enzyme Pc AFB1A and Gm The optimal reaction temperature of AFB1A Water baths at 25°C, 35°C, 45°C, 55°C, 65°C, 75°C, and 85°C were used to investigate the ability of recombinant aflatoxin B1-degrading enzyme to degrade AFB1 standards at different temperatures. Three replicates were set up at each temperature, and the total reaction volume was 500 µL. Control and experimental groups were set up separately. The control group consisted of 250 µL of phosphate buffer, 50 µL of 1000 ppb AFB1 standard, and 200 µL of blank supernatant (supernatant from yeast expression without the AFB1 degrading enzyme gene). The experimental group consisted of 250 µL of phosphate buffer, 50 µL of 1000 ppb AFB1 standard, and 200 µL of recombinant aflatoxin B1-degrading enzyme. The reaction mixtures were shaken in a water bath at 180 rpm for 10 hours. Subsequently, the samples were diluted as required, and the residual AFB1 toxin content in the samples was determined using an AFB1 enzyme-linked immunosorbent assay kit. The amount of recombinant aflatoxin B1-degrading enzyme was calculated. Pc AFB1A and Gm The degradation rate of AFB1 by AFB1A at different temperatures.

[0037] The results are as follows 图1 , recombinant aflatoxin B1 degrading enzyme Pc AFB1A and Gm The optimal temperature for AFB1A is 35℃.

[0038] Example 4: Recombinant aflatoxin B1 degrading enzyme Pc AFB1A and Gm The optimal reaction pH of AFB1A Buffer solutions with initial pH values ​​of 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, and 9.0 were selected to investigate the degradation capacity of recombinant aflatoxin B1-degrading enzyme against AFB1 standards at different pH values. The total reaction volume was 500 µL, with control and experimental groups set up. The control group consisted of 50 µL of 1000 ppb AFB1 standard, 200 µL of empty supernatant (supernatant from yeast expression without the AFB1-degrading enzyme gene), and 250 µL of buffer solutions at different pH values. The experimental group consisted of 50 µL of 1000 ppb AFB1 standard, 200 µL of recombinant aflatoxin B1-degrading enzyme, and 250 µL of buffer solutions at different pH values. The reaction solutions from the different groups were placed in a shaker at 35°C and 180 rpm for 10 h. Subsequently, the samples were diluted as required, and the residual AFB1 toxin content in the samples was determined using an AFB1 enzyme-linked immunosorbent assay kit. The amount of recombinant aflatoxin B1-degrading enzyme was calculated. Pc AFB1A and Gm The degradation rate of AFB1 by AFB1A under different pH conditions.

[0039] The results are as follows 图2 , recombinant aflatoxin B1 degrading enzyme Pc AFB1A and Gm The optimal pH for AFB1A is 6.0.

[0040] Example 5: Recombinant aflatoxin B1 degrading enzyme Pc AFB1A and Gm Optimal response time of AFB1A The ability of recombinant aflatoxin B1-degrading enzyme to degrade AFB1 standards was investigated using enzymatic digestion times of 24, 30, 36, 42, and 48 hours. A total reaction volume of 500 µL was used, with control and experimental groups set up. The control group consisted of 250 µL of phosphate buffer, 50 µL of a 1000 ppb AFB1 standard, and 200 µL of blank supernatant (supernatant from yeast expressing the AFB1-degrading enzyme gene). The experimental group consisted of 250 µL of phosphate buffer, 50 µL of a 1000 ppb AFB1 standard, and 200 µL of recombinant aflatoxin B1-degrading enzyme. The reactions were incubated at 35°C on a shaker at 180 rpm for 24, 30, 36, 42, and 48 hours, respectively. Subsequently, the sample was diluted as required and the residual AFB1 toxin content in the sample was determined using an AFB1 enzyme-linked immunosorbent assay kit to calculate the recombinant aflatoxin B1 degrading enzyme. Pc AFB1A and Gm The degradation rate of AFB1 after incubation of AFB1A with AFB1 for different time periods.

[0041] Results are as follows 图3 recombinant aflatoxin B1-degrading enzyme Pc AFB1A and Gm The optimal reaction length of AFB1A and AFB1A was 36 h.

[0042] Example 6: Recombinant aflatoxin B1-degrading enzyme Pc AFB1A and Gm AFB1 removal efficiency of AFB1A and AFB1A The experiment was divided into two groups: a control group containing 250 μL of phosphate buffer, 50 μL of 1000 ppb AFB1 standard, and 200 μL of empty supernatant (supernatant of yeast expression without AFB1-degrading enzyme gene), and an experimental group containing 250 μL of phosphate buffer, 50 μL of 1000 ppb AFB1 standard, and 200 μL of single enzyme / complex enzyme enzyme solution (volume ratio of double complex enzymes was 1:1, and the concentration of AFB1-degrading enzyme Pc AFB1A was used at a concentration of 2.48 mg / mL, and the concentration of AFB1-degrading enzyme Gm AFB1A was used at a concentration of 2.12 mg / mL, and 100 μL of each enzyme) was used. The control group and experimental group samples were incubated at 35°C in a 180 rpm shaker for 36 h, and then diluted as required. The residual AFB1 toxin content in the samples was determined using an AFB1 enzyme-linked immunoassay quantitative test kit, and the AFB1 removal efficiency of AFB1A and AFB1A was calculated, respectively. Pc AFB1A and Gm AFB1 degradation rates of AFB1A and AFB1A when used alone and together. The operation process was strictly in accordance with the kit instructions. All tests were repeated three times.

[0043] Results are as follows 图4 recombinant aflatoxin B1-degrading enzyme Pc AFB1A and Gm The AFB1 degradation rates of AFB1A and AFB1A were 36.58% and 34.93%, respectively, and the AFB1 degradation rate when used together was up to 41.18%.

[0044] Example 7: Recombinant aflatoxin B1-degrading enzyme Pc AFB1A and Gm AFB1 removal efficiency of AFB1A and AFB1A in moldy corn The experiment was divided into two groups: a control group containing 0.5 g of moldy corn (AFB1 content was 100 ppb), 1.8 mL of pH=6 buffer, and 200 μL of empty supernatant (supernatant of yeast expression without AFB1-degrading enzyme gene), and an experimental group containing 0.5 g of moldy corn, 1.8 mL of pH=6 buffer, and 200 μL of single enzyme / complex enzyme enzyme solution (volume ratio of double complex enzymes was 1:1, and the concentration of AFB1-degrading enzyme Pc AFB1A was used at a concentration of 2.48 mg / mL, and the degrading enzyme Gm The concentration of AFB1A was 2.12 mg / mL, and each enzyme was 100 μL. The control and experimental samples were incubated in a shaker at 35°C and 180 rpm for 36 h, and then diluted as required. The residual AFB1 toxin content in the samples was determined using an AFB1 enzyme-linked immunosorbent assay kit, and the recombinant aflatoxin B1 degrading enzyme was calculated. Pc AFB1A and Gm Degradation rates of AFB1 in moldy corn when AFB1A was used alone and in combination. The procedures were performed strictly according to the kit instructions. All experiments were repeated three times.

[0045] The results are as follows 图5 , recombinant aflatoxin B1 degrading enzyme Pc AFB1A and Gm The removal efficiencies of AFB1A on AFB1 in moldy corn were 19.43% and 18.65%, respectively. When used synergistically, the degradation rate of AFB1 was 25.17%.

[0046] The present invention provides two recombinant aflatoxin B1 degrading enzymes Pc AFB1A and Gm AFB1A, whose amino acid sequence is different from the amino acid sequence of the currently characterized aflatoxin B1 degrading enzyme, is a new aflatoxin B1 degrading enzyme that can enrich the existing AFB1 bioenzyme degradation resource library and provide a new option for AFB1 bioenzyme degradation. Pc AFB1A and Gm AFB1A can degrade AFB1 under mild conditions (pH=6.0, 35℃), and has a promoting effect on the degradation of AFB1 when used in combination with equal volumes. This enriches the AFB1 biological detoxification resource library, contributes to the development of efficient AFB1 detoxification complex enzymes, and shows potential application prospects in detoxifying AFB1-contaminated food and feed.

[0047] 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. An aflatoxin B1 degrading enzyme, characterized in that: The aflatoxin B1 degrading enzyme is a degrading enzyme PC AFB1A or degradation enzyme Gm AFB1A, a degrading enzyme PC The amino acid sequence of AFB1A is shown in SEQ ID NO.

1. Gm The amino acid sequence of AFB1A is shown in SEQ ID NO.

2.

2. The gene encoding the aflatoxin B1 degrading enzyme according to claim 1, characterized in that: Degradation enzymes PC The nucleotide sequence of the AFB1A coding gene is shown in SEQ ID NO.

3. Gm The nucleotide sequence of the AFB1A encoding gene is shown in SEQ ID NO.

4.

3. A recombinant vector comprising the encoding gene according to claim 2.

4. The recombinant vector according to claim 3, characterized in that: The recombinant vector is pPICZαA- PC AFB1A or pPICZαA- Gm AFB1A.

5. A recombinant strain comprising the encoding gene according to claim 2.

6. The method for preparing the aflatoxin B1 degrading enzyme according to claim 1, characterized in that: The steps are: (1) Transforming the recombinant vector described in claim 4 into Pichia pastoris X-33 cells to obtain a recombinant strain; (2) The seed solution of the recombinant strain was inoculated into BMGY medium at a ratio of 2% and cultured for 24 h. After centrifugation, the bacterial pellet was resuspended in BMMY induction medium and methanol was added every 24 h for 3 days. (3) After the culture is completed, centrifugation is performed and the resulting supernatant is aflatoxin B1 degrading enzyme; The recombinant vector is pPICZαA- PC When AFB1A is expressed, the supernatant is the degrading enzyme PC AFB1A; the recombinant vector is pPICZαA- Gm When AFB1A is present, the supernatant is the degrading enzyme Gm AFB1A.

7. A detoxification enzyme preparation, characterized in that Includes any of the following situations: a. Degradation enzymes PC AFB1A is the active ingredient; b. Degradation enzymes Gm AFB1A is the active ingredient; c. Degradation enzymes PC AFB1A and degrading enzymes Gm AFB1A is the active ingredient.

8. The detoxifying enzyme preparation according to claim 7, wherein: The degradative enzyme PC The amino acid sequence of AFB1A is shown in SEQ ID NO.

1. Gm The amino acid sequence of AFB1A is shown in SEQ ID NO. 2; the degradation enzyme in case a PC The concentration of AFB1A was 2.48 mg / mL, and the degradation enzyme in case b Gm The concentration of AFB1A was 2.12 mg / mL; in case c, the degradation enzyme PC AFB1A and degrading enzymes Gm The volume ratio of AFB1A when used in combination was 1:1, and the concentrations were 2.48 mg / mL and 2.12 mg / mL, respectively.

9. Use of the aflatoxin B1 degrading enzyme according to claim 1, the encoding gene according to claim 2, the recombinant vector according to claim 4, the recombinant strain according to claim 5, or the detoxification enzyme preparation according to claim 7 in mycotoxin detoxification.

10. The use according to claim 9, characterized in that: The mycotoxin is aflatoxin B1 in moldy corn; the detoxification conditions are 35° C. and pH=6.0 for 36 hours.

Images