Method for efficiently expressing aflatoxin degrading enzyme by bacillus subtilis

Abstract

The invention provides a method for efficiently expressing aflatoxin degrading enzyme by bacillus subtilis, and relates to the field of microbial fermentation and application thereof, and the method comprises the following steps: selecting a liquid culture medium which comprises the following components in parts by mass: 1-2 parts of a carbon source, 0.5-1 part of a nitrogen source, 0.1-0.3 part of inorganic salt and 0.01-0.05 part of vitamin; an expression vector containing an aflatoxin degrading enzyme coding gene is introduced into bacillus subtilis to obtain a recombinant strain, and the aflatoxin degrading enzyme coding gene is optimized by a bacillus subtilis codon optimization technology. The bacillus subtilis efficiently expresses the aflatoxin degrading enzyme, the expression level and enzyme activity of the aflatoxin degrading enzyme are remarkably improved by optimizing culture medium components, fermentation conditions and a gene codon optimization technology, the higher degrading enzyme yield is achieved, and the bacillus subtilis has higher stability and degradation efficiency; and the method shows a more excellent application effect in food safety treatment.

Description

Technical Field

[0001] The present invention relates to microbial fermentation and its application field, and specifically to a method for efficiently expressing aflatoxin degrading enzyme by Bacillus subtilis. Background Art

[0002] Currently, the production of aflatoxin-degrading enzymes primarily relies on microbial fermentation technology. Various microorganisms, such as fungi, bacteria, and their engineered strains, are used to produce aflatoxin-degrading enzymes. Through transgenic technology, the genes encoding these enzymes are introduced into microorganisms, enabling efficient synthesis of the enzymes and significant degradation of aflatoxins. Bacillus subtilis, as an important industrial microorganism, is widely used in the production of enzyme preparations due to its rapid growth, strong enzyme production capacity, and resistance to environmental stress. Furthermore, Bacillus subtilis possesses strong fermentation capacity and a sophisticated expression system, enabling efficient expression of exogenous proteins, making it an ideal host for aflatoxin-degrading enzymes. However, research on the efficient expression of aflatoxin-degrading enzymes in Bacillus subtilis remains in its exploratory stages.

[0003] Currently, several studies have employed various genetically engineered strains of Bacillus subtilis to produce aflatoxin-degrading enzymes, achieving promising degradation results. These studies have intensively explored strain selection, gene transfer, and optimization of fermentation conditions, achieving considerable progress. However, existing technologies still have room for improvement in terms of enzyme expression, degradation rate, and enzyme stability, hindering their effectiveness in practical applications.

[0004] Despite extensive research efforts devoted to developing efficient aflatoxin-degrading enzymes, existing technologies still suffer from a significant drawback: suboptimal enzyme expression. While existing Bacillus subtilis high-efficiency expression systems can achieve enzyme synthesis under certain conditions, they are often limited by factors such as cell growth, protein folding, and enzyme activity maintenance. In particular, low enzyme expression levels result in poor performance in practical applications, making them ineffective for large-scale food safety management. Summary of the Invention

[0005] In response to the deficiencies of the existing technology, the present invention provides a method for efficiently expressing aflatoxin-degrading enzyme in Bacillus subtilis. The technical problem to be solved by this invention is: how to achieve efficient expression of aflatoxin-degrading enzyme, enhance degradation effect and long-term stability through Bacillus subtilis codon optimization, fermentation culture condition optimization, induction system control and enzyme purification technology.

[0006] To achieve the above objectives, the present invention is implemented through the following technical scheme: a method for efficiently expressing aflatoxin degrading enzyme by Bacillus subtilis, comprising: S1. selecting a liquid culture medium, the mass ratios of the components of the liquid culture medium being: 1 to 2 parts of carbon source, 0.5 to 1 part of nitrogen source, 0.1 to 0.3 parts of inorganic salt, and 0.01 to 0.05 parts of vitamin.

[0007] S2. An expression vector containing a gene encoding an aflatoxin degrading enzyme is introduced into Bacillus subtilis to obtain a recombinant strain, wherein the gene encoding the aflatoxin degrading enzyme is optimized using Bacillus subtilis codon optimization technology to increase the expression level and activity of the enzyme.

[0008] S3. inoculating the recombinant strain into the liquid culture medium, and fermenting and culturing the liquid culture medium at a temperature of 28° C. to 32° C. for 12 to 48 hours.

[0009] S4. During the fermentation process, isopropyl-β-D-thiogalactoside is added as an inducer, the concentration of the isopropyl-β-D-thiogalactoside is 0.1 mM to 3 mM, and the induction time of the isopropyl-β-D-thiogalactoside is 6 to 8 hours.

[0010] S5. After the fermentation culture is completed, the fermentation broth is collected and the fermentation broth is post-processed to obtain a high-purity aflatoxin-degrading enzyme.

[0011] Preferably, the Bacillus subtilis codon optimization technology is specifically to improve the expression efficiency of the aflatoxin degrading enzyme encoding gene by adjusting the codon sequence of the aflatoxin degrading enzyme encoding gene, and the fermentation culture is carried out in a 5L fermentor, the fermentation culture has an aeration volume of 0.5vvm, a stirring rate of 250rpm, and the dissolved oxygen is maintained above 30%.

[0012] Preferably, the post-treatment includes centrifugation, filtration and purification. The specific steps of the post-treatment include:

[0013] S51. The centrifugation speed was 8000 rpm and the centrifugation time was 10 minutes. During the centrifugation process, the temperature was lowered to 4°C at a rate of 1°C / min to ensure stable enzyme activity and reduce degradation;

[0014] S52. The fermentation broth after the centrifugation was filtered to obtain a filtrate, and 50 ml to 100 ml of a salt solution was added during the filtration process to maintain the stability of the target enzyme and avoid enzyme inactivation due to membrane filtration;

[0015] S53. The filtration process uses a 0.22 micron filter membrane to remove cell residues and impurities in the fermentation broth.

[0016] Preferably, the purification treatment uses affinity chromatography to purify the filtrate to obtain the aflatoxin degrading enzyme, the affinity chromatography uses agarose gel as an affinity matrix, and the elution conditions of the purification step are PBS buffer containing 250mM imidazole and pH 7.4, which can purify the degrading enzyme from the fermentation broth to a purity of more than 90%.

[0017] Preferably, the catalytic reaction equation of the aflatoxin degrading enzyme is:

[0018]

[0019] in, Aflatoxin, For water, As the degradation product, As a by-product.

[0020] Preferably, the catalytic reaction is carried out at a pH of 7.0 and a temperature of The reaction rate is .

[0021] Preferably, the aflatoxin degrading enzyme is stored at -80°C in a PBS buffer containing 20% ​​glycerol, and the storage period is ≥6 months for long-term storage and subsequent use.

[0022] The present invention provides a method for efficiently expressing aflatoxin-degrading enzyme using Bacillus subtilis. The method has the following beneficial effects:

[0023] The present invention significantly increases the expression and activity of an aflatoxin-degrading enzyme by optimizing the gene expression system of Bacillus subtilis. Codon optimization technology increases the enzyme's expression efficiency, ensuring efficient synthesis and high enzyme activity during the fermentation process. Optimizing the gene expression system of Bacillus subtilis not only enhances the ability to degrade aflatoxin but also provides a feasible technical solution for large-scale production.

[0024] This Bacillus subtilis strain efficiently expresses an aflatoxin-degrading enzyme, optimizing the enzyme's purification and storage processes to ensure high purity and stability. The fermentation broth is purified through centrifugation, filtration, and affinity chromatography, ensuring the high efficiency and long-term shelf life of the final enzyme preparation. Storage at -80°C and the addition of glycerol for protection extend the enzyme's shelf life, ensuring its stability in practical applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is a flow chart of the expression of aflatoxin-degrading enzymes by Bacillus subtilis;

[0026] Figure 2 This is a flow chart for the purification of aflatoxin-degrading enzymes;

[0027] Figure 3 Schematic diagram of the storage conditions of aflatoxin-degrading enzymes. DETAILED DESCRIPTION

[0028] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0029] Example 1

[0030] like Figure 1-3 As shown, the present embodiment provides a method for efficiently expressing an aflatoxin-degrading enzyme in Bacillus subtilis, comprising: S1. selecting a liquid culture medium, wherein the mass ratios of the components of the liquid culture medium are: 1.5 parts carbon source, 0.75 parts nitrogen source, 0.2 parts inorganic salt, and 0.03 parts vitamin. The carbon source is glucose with a glucose concentration of 30 g / L, the nitrogen source is amino acids with an amino acid concentration of 10 g / L, the concentration of the inorganic salts, including sodium salts, is 2 g / L, and the pH value of the liquid culture medium is 7.0.

[0031] S2. An expression vector containing a gene encoding an aflatoxin-degrading enzyme was introduced into Bacillus subtilis to obtain a recombinant strain. The gene encoding the aflatoxin-degrading enzyme was optimized using Bacillus subtilis codon optimization technology to increase the expression level and activity of the enzyme. Specifically, the Bacillus subtilis codon optimization technology involves adjusting the codon sequence of the gene encoding the aflatoxin-degrading enzyme to increase the expression efficiency of the gene encoding the aflatoxin-degrading enzyme. Fermentation was performed in a 5L fermentor with an aeration rate of 0.5 vvm and an agitation rate of 250 rpm.

[0032] S3. The recombinant strain was inoculated into a liquid culture medium and fermented at 28°C for 30 hours.

[0033] S4. During the fermentation process, isopropyl-β-D-thiogalactoside was added as an inducer. The concentration of isopropyl-β-D-thiogalactoside was 0.1 mM to 3 mM, and the induction time of isopropyl-β-D-thiogalactoside was 7 hours.

[0034] S5. After the fermentation is complete, the fermentation broth is collected and subjected to post-processing to obtain a high-purity aflatoxin-degrading enzyme. Post-processing includes centrifugation, filtration, and purification. The specific steps of post-processing include:

[0035] S51. The rotation speed during the centrifugation is 8000 rpm and the centrifugation time is 10 minutes. During the centrifugation, the temperature is lowered to 4°C at a rate of 1°C / min.

[0036] S52. Filter the fermentation broth after centrifugation to obtain a filtrate, and add 75 ml of salt solution during the filtration process.

[0037] S53. Filtration treatment uses a 0.22 micron filter membrane to remove cell residues and impurities in the fermentation broth.

[0038] The aflatoxin-degrading enzyme is purified from the filtrate using affinity chromatography using agarose gel as the affinity matrix. Elution conditions for the purification step are PBS buffer containing 250 mM imidazole, pH 7.4. The enzyme can be purified from the fermentation broth to a purity exceeding 90%. The aflatoxin-degrading enzyme is stored at -80°C in PBS buffer containing 20% ​​glycerol for 6 months or longer.

[0039] The catalytic reaction equation of aflatoxin degrading enzyme is:

[0040]

[0041] in, Aflatoxin, For water, As the degradation product, As a by-product. The catalytic reaction is carried out at a pH of 7.0 and a temperature of The reaction rate is .

[0042] Enzyme activity:

[0043] The enzyme activity of the mean component culture medium was higher, and the aflatoxin degradation rate reached 75%. Under this condition, the enzyme expression effect was better.

[0044] Degradation effect:

[0045] Under these conditions, Bacillus subtilis can effectively express and activate aflatoxin-degrading enzymes.

[0046] Enzyme stability:

[0047] Under long-term storage, the enzyme produced by the mean culture medium has good stability and the enzyme activity remains at a high level.

[0048] Example 2

[0049] This embodiment is based on the first embodiment, and adopts the culture medium with low component ratio conditions for culturing, so that Bacillus subtilis can efficiently express and maintain enzyme activity under low nutrient conditions.

[0050] S1. Select a liquid culture medium containing the following components by weight: 1 part carbon source, 0.5 part nitrogen source, 0.1 part inorganic salt, and 0.01 part vitamin. The carbon source is glucose at a glucose concentration of 20 g / L, the nitrogen source is amino acids at a concentration of 5 g / L, and the inorganic salts, including sodium salts, are at a concentration of 1 g / L. The pH of the liquid culture medium is 6.8.

[0051] S2. An expression vector containing a gene encoding an aflatoxin-degrading enzyme was introduced into Bacillus subtilis to obtain a recombinant strain. The gene encoding the aflatoxin-degrading enzyme was optimized using Bacillus subtilis codon optimization technology to increase the expression level and activity of the enzyme. Specifically, the Bacillus subtilis codon optimization technology involves adjusting the codon sequence of the gene encoding the aflatoxin-degrading enzyme to increase the expression efficiency of the gene encoding the aflatoxin-degrading enzyme. Fermentation was performed in a 5L fermentor with an aeration rate of 0.5 vvm and an agitation rate of 250 rpm.

[0052] S3. The recombinant strain was inoculated into a liquid culture medium and fermented at 28°C for 24 hours.

[0053] S4. During the fermentation process, isopropyl-β-D-thiogalactoside is added as an inducer. The concentration of isopropyl-β-D-thiogalactoside is 0.1 mM to 3 mM, and the induction time of isopropyl-β-D-thiogalactoside is 6 hours.

[0054] S5. After the fermentation is complete, the fermentation broth is collected and subjected to post-processing to obtain a high-purity aflatoxin-degrading enzyme. Post-processing includes centrifugation, filtration, and purification. The specific steps of post-processing include:

[0055] S51. The rotation speed during the centrifugation is 8000 rpm and the centrifugation time is 10 minutes. During the centrifugation, the temperature is lowered to 4°C at a rate of 1°C / min.

[0056] S52. Filter the fermentation broth after centrifugation to obtain a filtrate, and add 75 ml of salt solution during the filtration process.

[0057] S53. Filtration treatment uses a 0.22 micron filter membrane to remove cell residues and impurities in the fermentation broth.

[0058] The aflatoxin-degrading enzyme is purified from the filtrate using affinity chromatography using agarose gel as the affinity matrix. Elution conditions for the purification step are PBS buffer containing 250 mM imidazole, pH 7.4. The enzyme can be purified from the fermentation broth to a purity exceeding 90%. The aflatoxin-degrading enzyme is stored at -80°C in PBS buffer containing 20% ​​glycerol for 6 months or longer.

[0059] The catalytic reaction equation of aflatoxin degrading enzyme is:

[0060]

[0061] in, Aflatoxin, For water, As the degradation product, As a by-product. The catalytic reaction is carried out at a pH of 7.0 and a temperature of The reaction rate is .

[0062] Enzyme activity:

[0063] It was determined that the expressed aflatoxin-degrading enzyme exhibited high activity under minimal culture medium conditions, proving that Bacillus subtilis can efficiently express and maintain enzyme activity under low nutrient conditions.

[0064] Degradation effect:

[0065] In the degradation experiment of aflatoxin by the purified enzyme, a degradation rate of 80% was shown, indicating that the enzyme has a strong degradation ability.

[0066] Enzyme stability:

[0067] After long-term storage, the activity of the enzyme is stable, indicating that the method of the present invention not only increases the yield of aflatoxin-degrading enzyme, but also ensures its long-term effectiveness.

[0068] Example 3

[0069] Based on Example 1, this example uses a culture medium with a high component ratio to perform culture, demonstrating that under nutrient-rich conditions, Bacillus subtilis can efficiently express and maintain enzyme activity.

[0070] S1. Select a liquid culture medium containing the following components by mass: 2 parts carbon source, 1 part nitrogen source, 0.3 parts inorganic salts, and 0.05 parts vitamins. The carbon source is glucose at a glucose concentration of 40 g / L, the nitrogen source is amino acids at a concentration of 15 g / L, and the inorganic salts, including sodium salts, are at a concentration of 3 g / L. The pH of the liquid culture medium is 7.0.

[0071] S2. An expression vector containing a gene encoding an aflatoxin-degrading enzyme was introduced into Bacillus subtilis to obtain a recombinant strain. The gene encoding the aflatoxin-degrading enzyme was optimized using Bacillus subtilis codon optimization technology to increase the expression level and activity of the enzyme. Specifically, the Bacillus subtilis codon optimization technology involves adjusting the codon sequence of the gene encoding the aflatoxin-degrading enzyme to increase the expression efficiency of the gene encoding the aflatoxin-degrading enzyme. Fermentation was performed in a 5L fermentor with an aeration rate of 0.5 vvm and an agitation rate of 250 rpm.

[0072] S3. The recombinant strain was inoculated into liquid culture medium and fermented at 28°C for 36 hours.

[0073] S4. During the fermentation process, isopropyl-β-D-thiogalactoside was added as an inducer. The concentration of isopropyl-β-D-thiogalactoside was 0.1 mM to 3 mM, and the induction time of isopropyl-β-D-thiogalactoside was 16 hours.

[0074] S5. After the fermentation is complete, the fermentation broth is collected and subjected to post-processing to obtain a high-purity aflatoxin-degrading enzyme. Post-processing includes centrifugation, filtration, and purification. The specific steps of post-processing include:

[0075] S51. The rotation speed during the centrifugation is 8000 rpm and the centrifugation time is 10 minutes. During the centrifugation, the temperature is lowered to 4°C at a rate of 1°C / min.

[0076] S52. Filter the fermentation broth after centrifugation to obtain a filtrate, and add 75 ml of salt solution during the filtration process.

[0077] S53. Filtration treatment uses a 0.22 micron filter membrane to remove cell residues and impurities in the fermentation broth.

[0078] The aflatoxin-degrading enzyme is purified from the filtrate using affinity chromatography using agarose gel as the affinity matrix. Elution conditions for the purification step are PBS buffer containing 250 mM imidazole, pH 7.4. The enzyme can be purified from the fermentation broth to a purity exceeding 90%. The aflatoxin-degrading enzyme is stored at -80°C in PBS buffer containing 20% ​​glycerol for 6 months or longer.

[0079] The catalytic reaction equation of aflatoxin degrading enzyme is:

[0080]

[0081] in, Aflatoxin, For water, As the degradation product, As a by-product. The catalytic reaction is carried out at a pH of 7.0 and a temperature of The reaction rate is .

[0082] Enzyme activity:

[0083] It was determined that the expressed aflatoxin-degrading enzyme exhibited high activity under maximum culture medium conditions, proving that under nutrient-rich conditions, Bacillus subtilis can efficiently express and maintain enzyme activity.

[0084] Degradation effect:

[0085] In the degradation experiment of aflatoxin by the purified enzyme, a degradation rate of 83% was shown. The enzyme has strong degradation ability and is suitable for practical application.

[0086] Enzyme stability:

[0087] After long-term storage, the activity of the enzyme is stable. The preparation method of the enzyme under these conditions increases the yield of aflatoxin-degrading enzyme and also ensures its long-term effectiveness.

[0088] Example 4

[0089] Different from Example 1, this example shows that the data range in the preparation method provides the optimal process parameters for the efficient expression of aflatoxin degrading enzyme by comparing with a high-ratio aflatoxin degrading enzyme preparation method outside the range.

[0090] 1. Experimental Preparation:

[0091] Culture medium components

[0092] Mean within culture method:

[0093] Carbon source: 1.5 parts glucose = 30g / L.

[0094] Nitrogen source: 0.75 parts of amino acid = 10g / L.

[0095] Inorganic salt: 0.2 parts of inorganic salt containing sodium salt = 2g / L.

[0096] Vitamins: 0.03 parts of vitamins = 0.03g / L.

[0097] pH value: 7.0.

[0098] High proportion components:

[0099] Carbon source: 3 parts glucose = 60g / L.

[0100] Nitrogen source: 1.5 parts of amino acid = 20g / L.

[0101] Inorganic salt: 0.3 parts of inorganic salt containing sodium salt = 3g / L.

[0102] Vitamins: 0.05 parts of vitamins = 0.05g / L.

[0103] pH value: 7.5.

[0104] Experimental Materials:

[0105] Bacillus subtilis expression vector containing the gene encoding aflatoxin-degrading enzyme.

[0106] Isopropyl-β-D-thiogalactopyranoside, the concentrations were set to 2 mM and 4 mM, respectively.

[0107] Other experimental materials: PBS buffer, glycerol, etc.

[0108] 2. Preparation of bacterial strains:

[0109] Genetically engineered Bacillus subtilis was selected as the host, and the optimized aflatoxin degrading enzyme gene vector was introduced into the Bacillus subtilis using the electroporation method to obtain a recombinant strain.

[0110] The successfully transformed strains were screened out by the culture medium plate screening method.

[0111] 3. Fermentation culture:

[0112] Vaccination:

[0113] The selected recombinant Bacillus subtilis was inoculated into two culture media respectively, with the inoculation amount being 1% of the volume of the culture medium.

[0114] Fermentation conditions:

[0115] Temperature: 28℃.

[0116] pH value: adjusted to 7.0 and 7.5 respectively.

[0117] Stirring rate: 250 rpm.

[0118] Dissolved oxygen content: maintained above 30%.

[0119] Cultivation time: 30 hours.

[0120] Induction:

[0121] After culturing for 23 hours, 2 mM and 4 mM isopropyl-β-D-thiogalactoside were added, respectively, and the culture was continued for 7 hours.

[0122] 4. Enzyme extraction and purification:

[0123] Collect the fermentation broth:

[0124] After the fermentation culture is completed, the culture broth is collected and the cell debris is removed by centrifugation.

[0125] Enzyme purification:

[0126] The enzyme was purified by affinity chromatography using agarose gel as the affinity matrix.

[0127] During the purification process, PBS buffer is used to elute the aflatoxin-degrading enzyme, and the purity of the purified enzyme should reach more than 90%.

[0128] 5. Enzyme activity assay:

[0129] Enzyme activity assay method:

[0130] Standard enzyme activity assays were used to assess the activity of the purified enzymes.

[0131] In the experiment, the concentration of degraded aflatoxin was measured at different time points and the degradation effect of the enzyme was calculated.

[0132] Experimental data:

[0133] Mean fraction culture medium:

[0134] Within 48 hours, the purified enzyme could degrade 100 μg / mL of aflatoxin to 25 μg / mL, with a degradation rate of 75%.

[0135] High ratio component culture medium:

[0136] Within 48 hours, the purified enzyme could degrade 100 μg / mL of aflatoxin to 35 μg / mL, with a degradation rate of 65%.

[0137] 6. Enzyme stability and long-term storage:

[0138] Stability test:

[0139] The purified aflatoxin-degrading enzyme was aliquoted and stored at -80°C using PBS buffer containing 20% ​​glycerol as the protective solution.

[0140] Check enzyme activity regularly.

[0141] Experimental data:

[0142] Average component culture medium enzyme: During the 6-month storage period, the enzyme activity remains above 72%.

[0143] High ratio of component culture medium enzyme: within 6 months of storage period, the enzyme activity remains above 60%.

[0144] 7. Experimental results and analysis:

[0145] Enzyme activity:

[0146] The enzyme activity of the mean component culture medium was higher, and the aflatoxin degradation rate reached 75%. Under this condition, the enzyme expression effect was better.

[0147] The enzyme activity of the high-proportion component culture medium is low, and the aflatoxin degradation rate is only 65%. The excessive carbon and nitrogen sources lead to the inhibition of enzyme synthesis or excessive synthesis, resulting in poor enzyme folding and stability.

[0148] Degradation effect:

[0149] The degradation effect of the mean component culture medium was better, indicating that Bacillus subtilis could more effectively express and activate aflatoxin-degrading enzymes under this condition.

[0150] The degradation effect of high-ratio component culture medium is poor, and excess nutrition has a negative impact on cell growth and protein folding process.

[0151] Enzyme stability:

[0152] Under long-term storage, the enzyme produced by the mean component culture medium has good stability and the enzyme activity remains at a high level.

[0153] The enzymes produced by high-ratio component culture media have poor stability, and the enzyme activity decreases more significantly during long-term storage.

[0154] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and spirit of the invention, and that the scope of the invention is defined by the appended claims and their equivalents.

Claims

1. A method for efficiently expressing aflatoxin-degrading enzyme by Bacillus subtilis, characterized in that: include: S1. Select a liquid culture medium, wherein the mass ratios of the components of the liquid culture medium are: 1 to 2 parts carbon source, 0.5 to 1 part nitrogen source, 0.1 to 0.3 parts inorganic salts, and 0.01 to 0.05 parts vitamins; S2. An expression vector containing an aflatoxin degrading enzyme encoding gene is introduced into Bacillus subtilis to obtain a recombinant strain, wherein the aflatoxin degrading enzyme encoding gene is optimized using Bacillus subtilis codon optimization technology; S3. inoculating the recombinant strain into the liquid culture medium and fermenting at a temperature of 28°C to 32°C for 12 to 48 hours; S4 isopropyl -β-D-thiogalactoside is added as an inducer during the fermentation culture, the concentration of the isopropyl -β-D-thiogalactoside is 0.1mM to 3mM, the induction time of the isopropyl -β-D-thiogalactoside is 6 to 8 hours; S5. After the fermentation culture is completed, the fermentation broth is collected and the fermentation broth is post-processed to obtain a high-purity aflatoxin-degrading enzyme.

2. The method for efficiently expressing aflatoxin-degrading enzyme by Bacillus subtilis according to claim 1, characterized in that: The carbon source is glucose, the glucose concentration is 20g / L to 40g / L, the nitrogen source is amino acids, the amino acid concentration is 5g / L to 15g / L, the inorganic salt contains sodium salt at a concentration of 1g / L to 3g / L, and the pH value of the liquid culture medium is in the range of 6.8 to 7.

2.

3. The method for efficiently expressing aflatoxin-degrading enzyme by Bacillus subtilis according to claim 1, characterized in that: The Bacillus subtilis codon optimization technology specifically improves the expression efficiency of the aflatoxin degrading enzyme encoding gene by adjusting the codon sequence of the aflatoxin degrading enzyme encoding gene. The fermentation culture is carried out in a 5L fermenter with an aeration volume of 0.5 vvm and a stirring rate of 250 rpm.

4. The method for efficiently expressing aflatoxin-degrading enzyme by Bacillus subtilis according to claim 1, characterized in that: The post-processing includes centrifugation, filtration and purification. The specific steps of the post-processing include: S51. The centrifugal speed is 8000 rpm, the centrifugation time is 10 minutes, and the temperature is reduced to 4°C at a rate of 1°C / min during the centrifugation process; S52. The fermentation broth after the centrifugation is filtered to obtain a filtrate, and 50 ml to 100 ml of a salt solution is added during the filtration process; S53. The filtration process uses a 0.22 micron filter membrane.

5. The method for efficiently expressing aflatoxin-degrading enzyme by Bacillus subtilis according to claim 4, characterized in that: The purification treatment uses affinity chromatography to purify the filtrate to obtain the aflatoxin degrading enzyme. The affinity chromatography uses agarose gel as an affinity matrix. The elution condition of the purification step is PBS buffer containing 250mM imidazole and pH 7.

4.

6. The method for efficiently expressing aflatoxin-degrading enzyme by Bacillus subtilis according to claim 1, characterized in that: The catalytic reaction equation of the aflatoxin degrading enzyme is: in, Aflatoxin, For water, As the degradation product, As a by-product.

7. The method for efficiently expressing aflatoxin-degrading enzyme by Bacillus subtilis according to claim 6, characterized in that: The catalytic reaction is carried out at a pH of 7.0 and a temperature of The reaction rate is .

8. The method for efficiently expressing aflatoxin-degrading enzyme by Bacillus subtilis according to claim 1, characterized in that: The aflatoxin degrading enzyme is stored at -80°C in an environment containing PBS buffer containing 20% ​​glycerol, and the storage period is ≥6 months.

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