Aflatoxin oxidase mutant E188R-S210G-T418A as well as coding gene and application thereof

Abstract

The invention belongs to the technical field of feed biology, and particularly relates to an aflatoxin oxidase mutant as well as a coding gene and application thereof. According to the three-point combined mutant E188R-S210G-T418A provided by the invention, the enzyme activity is 177% of that of a female parent BlCotA; after water bath is carried out for 30 minutes at 80 DEG C, the enzyme activity is 68% of the initial enzyme activity, and the thermal stability is superior to that of a female parent BlCotA; after the mutant E188R-S210 G-T418A is used for treating raw milk for 1 hour, 2 hours and 4 hours, the residual quantity of the AFM1 is respectively reduced to 0.31 ng / mL, 0.12 ng / mL and 0.01 ng / mL; after corn steep liquor is treated by the mutant E188R-S210G-T418A, the degradation rates of AFB1, AFB2, AFG1 and AFG2 are respectively 93.71 percent, 93.95 percent, 96.79 percent and 97.58 percent.

Description

Technical Field

[0001] This invention belongs to the field of feed biotechnology, and specifically relates to an aflatoxin oxidase mutant, its encoding gene, and its applications. Background Technology

[0002] Aflatoxins are a class of mycotoxins, primarily produced by Aspergillus flavus and Aspergillus parasiticus, and are highly carcinogenic. Scientists have currently identified more than 20 aflatoxins, including aflatoxin B1 (AFB1), aflatoxin B2 (AFB2), aflatoxin G1 (AFG1), aflatoxin G2 (AFG2), aflatoxin M1 (AFM1), and aflatoxin M2 (AFM2). Among the aflatoxin family, AFB1 is the most toxic and carcinogenic, with a toxicity 100 times that of potassium cyanide, 75 times that of dimethylnitrosamine, and 68 times that of arsenic. Aflatoxins widely contaminate grains such as corn, wheat, and barley, as well as their processing byproducts, posing a significant risk to feed safety. Consuming aflatoxin-contaminated feed can lead to liver and intestinal damage, metabolic disorders, immunosuppression, and decreased production performance in livestock and poultry. Furthermore, aflatoxins in feed can transfer to livestock and poultry products (such as meat, eggs, and milk), threatening food safety.

[0003] Microbial enzymatic hydrolysis, which converts aflatoxin into non-toxic or low-toxic metabolites, is considered the most promising method for aflatoxin detoxification in feed. Compared to physical and chemical detoxification methods, microbial enzymatic hydrolysis offers advantages such as safety, high efficiency, and minimal impact on feed nutritional value. However, most currently discovered aflatoxin-degrading enzymes have low activity, hindering their large-scale industrial production and application. Patent 202411850569.7 discloses an aflatoxin oxidase, DnD-AFO, which exhibits higher activity and better thermal stability compared to the traditional wild-type oxidase AFO. Therefore, developing aflatoxin oxidase with high activity, strong stability, and wide applicability is crucial for overcoming the bottlenecks of existing enzymatic detoxification technologies and constructing a safe, efficient, and low-cost aflatoxin contamination control system. It also holds broad application prospects in the food industry, feed industry, and agricultural product safety. Summary of the Invention

[0004] To address the above problems, this invention proposes an aflatoxin oxidase mutant E188R-S210G-T418A, its encoding gene, and its applications.

[0005] The technical solution of this invention is implemented as follows: This invention proposes an aflatoxin oxidase mutant E188R-S210G-T418A, the amino acid sequence of which is shown in SEQ ID NO.2.

[0006] Preferably, the mutant E188R-S210G-T418A is an aflatoxin oxidase with the amino acid sequence shown in SEQ ID NO.1. Bl CotA was obtained by simultaneously mutating amino acids at positions 188, 210, and 418; specifically, glutamic acid at position 188 was mutated to arginine, serine at position 210 was mutated to glycine, and threonine at position 418 was mutated to alanine.

[0007] aflatoxin oxidase Bl The amino acid sequence of CotA is shown in SEQ ID NO.1 of the sequence listing, and the nucleotide sequence of the coding gene after codon optimization is shown in SEQ ID NO.3 of the sequence listing, which was synthesized by a commercial company.

[0008] This invention uses computational design to obtain three potential single-point positive mutants: glutamic acid at position 188 is mutated to arginine (mutant named E188R), serine at position 210 is mutated to glycine (mutant named S210G), and threonine at position 418 is mutated to alanine (mutant named T418A).

[0009] Secondly, the present invention provides a gene encoding an aflatoxin oxidase mutant E188R-S210G-T418A, which encodes the aforementioned aflatoxin oxidase mutant E188R-S210G-T418A; its nucleotide sequence is shown in SEQ ID NO.4.

[0010] Thirdly, the present invention also provides a recombinant expression vector containing the above-mentioned coding gene. The starting vector of the recombinant expression vector is specifically pET-31b.

[0011] Fourthly, the present invention provides a recombinant expression strain containing the above-mentioned recombinant expression vector. The starting strain of the recombinant expression strain is specifically *Escherichia coli* BL21.

[0012] Fifthly, the present invention provides a method for preparing the above-mentioned aflatoxin oxidase mutant E188R-S210G-T418A, comprising the following steps: 1) Using pET-31b as the starting vector, aflatoxin oxidase was inserted into the vector. Bl The gene encoding the CotA mutant E188R-S210G-T418A was transformed into Escherichia coli BL21 to obtain a recombinant strain; 2) Cultivate the recombinant strain to induce aflatoxin oxidase. Bl CotA mutant E188R-S210G-T418A expression.

[0013] 3) Recover and purify the expressed aflatoxin oxidase. Bl CotA mutant E188R-S210G-T418A protein.

[0014] In a sixth aspect, the present invention provides the application of the above-mentioned aflatoxin oxidase mutant E188R-S210G-T418A in removing aflatoxin from grains, feed, nuts, milk, corn steep liquor or herbal extracts.

[0015] Preferably, the aflatoxins mentioned above include at least one of aflatoxin B1, aflatoxin B2, aflatoxin G1, aflatoxin G2 and aflatoxin M1.

[0016] Preferably, 10-100 mg of aflatoxin oxidase mutant E188R-S210G-T418A is added to every 100 mL of corn steep liquor or milk.

[0017] The present invention has the following beneficial effects: This invention, through computational design, yielded three potential single-point positive mutants: E188R, S210G, and T418A, as well as a three-point combination mutant, E188R-S210G-T418A. The enzyme activities of these mutants are respectively those of the parent... Bl The residual enzyme activity of CotA was 134%, 125%, 137%, and 177%; after incubation at 80℃ for 10, 20, and 30 min, the residual enzyme activity of mutant E188R-S210G-T418A was 91%, 83%, and 68% of the initial enzyme activity, respectively, indicating better thermostability than the parent protein. Bl After treatment of raw milk with CotA mutant E188R-S210G-T418A for 1 h, 2 h, and 4 h, the residual AFM1 content decreased to 0.31 ng / mL, 0.12 ng / mL, and 0.01 ng / mL, respectively. After treatment of corn steep liquor with mutant E188R-S210G-T418A, the contents of AFB1, AFB2, AFG1, and AFG2 decreased to 13.4 ng / mL, 1.3 ng / mL, 1.5 ng / mL, and 0.3 ng / mL, respectively, and the degradation rates of AFB1, AFB2, AFG1, and AFG2 were 93.71%, 93.95%, 96.79%, and 97.58%, respectively. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 aflatoxin oxidase Bl SDS-PAGE electrophoresis results of CotA and its mutants.

[0020] Figure 2 aflatoxin oxidase Bl Comparison of the enzyme activities of CotA and its mutants.

[0021] Figure 3 The mutant E188R-S210G-T418A and the maternal parent Bl Comparison results of the thermal stability of CotA. Detailed Implementation

[0022] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0023] Unless otherwise specified, the experimental methods used in the following experimental examples are conventional methods; the materials and reagents used are commercially available unless otherwise specified.

[0024] The main experimental materials and reagents used in the embodiments of this invention are as follows: Escherichia coli expression vector pET-31b and Escherichia coli BL21 were purchased from Invitrogen; restriction endonucleases, DNA polymerases and DNA ligases were purchased from NEB; aflatoxin B1, aflatoxin B2, aflatoxin G1, aflatoxin G2 and aflatoxin M1 standards were purchased from Qingdao Pribang Biotechnology Co., Ltd.; and the QuickMutation™ gene site-directed mutagenesis kit was purchased from Shanghai Beyotime Biotechnology Co., Ltd.

[0025] Unless otherwise specified, all other biochemical reagents used in the examples are commercially available reagents, and the technical means used in the examples are conventional means used by those skilled in the art.

[0026] Example 1: Aflatoxin oxidase Bl Construction of CotA single-point mutant The aflatoxin oxidase used in this embodiment Bl The amino acid sequence of CotA is shown in SEQ ID NO.1 of the sequence listing, and the nucleotide sequence of the coding gene after codon optimization is shown in SEQ ID NO.3 of the sequence listing, which was synthesized by a commercial company.

[0027] Synthesized aflatoxin oxidase Bl The gene encoding CotA is linked to the pET-31b vector. NdeI and XhoI Between the restriction enzyme sites, the recombinant plasmid pET-31b- was obtained. Bl CotA.

[0028] Three potential positive mutants were obtained through computational design: glutamic acid at position 188 was mutated to arginine (mutant named E188R), serine at position 210 was mutated to glycine (mutant named S210G), and threonine at position 418 was mutated to alanine (mutant named T418A).

[0029] The three mutants described above were constructed according to the QuickMutation™ site-directed mutagenesis kit instructions. The two primers designed to construct the mutant E188R are as follows: Upstream primer E188R-F (SEQ ID NO.5): 5'-CGGCGGGCCGATATGATGTGC-3' Downstream primer E188R-R (SEQ ID NO.6): 5'-GCACATCATATCGGCCCGCCG-3' The two primers designed to construct the mutant S210G are as follows: Upstream primer S210G-F (SEQ ID NO.7): 5'-CTGTTTTATCCGGGCGGCCCGGA-3' Downstream primer S210G-R (SEQ ID NO.8): 5'-TCCGGGCCGCCCGGATAAAACAG-3' The two primers designed to construct the mutant T418A are as follows: Upstream primer T418A-F (SEQ ID NO.9): 5'-CCCGCGGCGCCCATCCGATTC-3' Downstream primer T418A-R (SEQ ID NO.10): 5'-GAATCGGATGGGCGCCGCGGG-3' With recombinant plasmid pET-31b- Bl Using CotA as a template, site-directed mutagenesis was performed via PCR. The reaction mixture was as follows: 36 μL ddH2O, 5 µL 10 × BeyoFusion™ Buffer, 5 µL dNTPmix, 1 µL BeyoFusion™ DNA Polymerase, and 1 µL recombinant plasmid pET-31b-Bl CotA, 1 µL upstream primer and 1 µL downstream primer; The PCR amplification conditions were as follows: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 30 s, 55℃ annealing for 30 s, 68℃ extension for 150 s, for 30 cycles; and 68℃ complete extension for 10 min.

[0030] After the PCR reaction was completed, 1 µL of DpnI was added to the reaction system, gently mixed, and then incubated in a 37°C water bath for 30 min to prepare the mutant plasmid.

[0031] The recombinant plasmid pET-31b- Bl CotA and the aforementioned constructed mutant plasmids pET-31b-E188R, pET-31b-S210G, and pET-31b-T418A were transformed into recombinant Escherichia coli BL21 to obtain the recombinant expression strain BL21 (pET-31b- Bl CotA), BL21 (pET-31b-E188R), BL21 (pET-31b-S210G) and BL21 (pET-31b-T418A).

[0032] Example 2: Aflatoxin oxidase Bl Construction of CotA three-point combination mutant Simultaneously, aflatoxin oxidase Bl The three-point combination mutant was named E188R-S210G-T418A, which was constructed by mutating glutamic acid at position 188 to arginine, serine at position 210 to glycine, and threonine at position 418 to alanine.

[0033] The construction method of mutant E188R-S210G-T418A is as follows: Based on the single-point mutant plasmid pET-31b-E188R constructed in Example 1, a two-point mutant plasmid was constructed by PCR using primers S210G-F and S210G-R. Further, based on the two-point mutant plasmid pET-31b-E188R-S210G, a three-point mutant plasmid pET-31b-E188R-S210G-T418A was constructed by PCR using primers T418A-F and T418A-R. The constructed mutant plasmid pET-31b-E188R-S210G-T418A was transformed into recombinant Escherichia coli BL21 to obtain the recombinant expression strain BL21 (pET-31b-E188R-S210G-T418A).

[0034] Example 3: Aflatoxin oxidase BlComparison of CotA expression and enzyme activity with its mutants The recombinant expression strain BL21 (pET-31b- Bl CotA), BL21 (pET-31b-E188R), BL21 (pET-31b-S210G), BL21 (pET-31b-T418A), and BL21 (pET-31b-E188R-S210G-T418A) were each inoculated into 5 mL LB liquid medium and activated overnight. Then, they were transferred at a 1:100 ratio to 300 mL Erlenmeyer flasks (500 mL each) and incubated at 37°C with shaking at 180 rpm until OD (out of control). 600 When the concentration was 0.6, 0.1 mM IPTG and 2 mM CuSO4 were added, and the mixture was cultured at 20℃ and 120 r / min for 20 h to induce the expression of the target protein.

[0035] Collect the fermentation broth, centrifuge at 12000 rpm for 30 min at 4℃, and discard the supernatant. Resuspend the cells in phosphate buffer (pH 7.0), centrifuge at 12000 rpm for 30 min at 4℃, discard the supernatant, and repeat the washing process three times. Resuspend the cells in binding buffer, sonicate them in an ice-water bath, centrifuge at 12000 rpm for 10 min at 4℃, and collect the supernatant to obtain the crude enzyme solution. Filter the crude enzyme solution through a 0.45 μm filter membrane and then perform chromatography using a nickel ion affinity column (Ni...). 2+ For purification steps such as equilibration, loading, and elution of hexahistine-labeled proteins (NTA), please refer to the Qiagen manual on purification of hexahistine-labeled proteins.

[0036] Purified aflatoxin oxidase Bl CotA and its mutant proteins were analyzed by SDS-PAGE electrophoresis. Results are as follows: Figure 1 As shown, lane 1 contains purified aflatoxin oxidase. Bl CotA migration band, lane 2 shows the purified mutant protein E188R migration band, lane 3 shows the purified mutant protein S210G migration band, lane 4 shows the purified mutant protein T418A migration band, lane 5 shows the purified mutant protein E188R-S210G-T418A migration band, aflatoxin oxidase. Bl CotA and its mutant proteins have the same molecular weight, approximately 59 kDa.

[0037] Solid aflatoxin B1 (AFB1) standard was dissolved in methanol to prepare a stock solution of 1.0 mg / mL. The experiment was conducted using the following 500 μL reaction system: 485 μL sodium phosphate buffer (100 mM, pH 7.0), 5 μL of the aflatoxin oxidase prepared above. BlCotA or its mutant protein (0.1 mg / mL), 10 μL AFB1 stock solution; after incubation in a 37℃ water bath for 0.5 h, 500 μL methanol was added to terminate the reaction. The AFB1 content in the reaction system was analyzed by high performance liquid chromatography. Bl The enzyme activity of CotA is defined as 100%, and the relative enzyme activity of the mutant is calculated.

[0038] The chromatographic conditions for high-performance liquid chromatography (HPLC) detection of AFB1 were as follows: column: Agilent C18 column, 4.6 mm × 250 mm × 5 μm; mobile phase: methanol-water (45:55); flow rate: 1 mL / min; column temperature: 30℃; injection volume: 20 μL; fluorescence detector set to excitation wavelength 360 nm and emission wavelength 440 nm. Acquisition time: 30 minutes.

[0039] The results are as follows Figure 2 As shown, the enzyme activities of mutants E188R, S210G, T418A, and E188R-S210G-T418A are respectively those of the parent... Bl CotA was 134%, 125%, 137%, and 177%.

[0040] Example 4: Mutant E188R-S210G-T418A and the maternal parent Bl CotA thermal stability test To determine the mutant E188R-S210G-T418A and the parent in this application Bl The thermal stability of CotA was compared between the mutant E188R-S210G-T418A and the maternal parent. Bl CotA was incubated in a water bath at 80°C for 10, 20, and 30 min, and then the mutant E188R-S210G-T418A and the maternal parent were measured using the method described in Example 3. Bl Remaining enzyme activity of CotA.

[0041] The results are as follows Figure 3 As shown, the parent Bl After CotA was incubated in a water bath at 80℃ for 10, 20, and 30 min, the remaining enzyme activities were 87%, 76%, and 52% of the initial enzyme activity, respectively. For the mutant E188R-S210G-T418A, after the same process, the remaining enzyme activities were 91%, 83%, and 68% of the initial enzyme activity, respectively. Therefore, the mutant E188R-S210G-T418A exhibits superior thermostability compared to the parent strain. Bl CotA.

[0042] Application Example 1: Aflatoxin Oxidase BlApplication of CotA and its mutant E188R-S210G-T418A in the removal of aflatoxin M1 (AFM1) from milk Add 100 mL of raw milk contaminated with AFM1 (AFM1 content of 0.64 ng / mL) to a 500 mL Erlenmeyer flask, and add 10 mg of aflatoxin oxidase prepared in Example 3. Bl CotA or mutant E188R-S210G-T418A enzyme protein were reacted at 25℃ for 1 h, 2 h and 4 h to determine the AFM1 residue in raw milk.

[0043] The determination method is as follows: Take 10 mL of raw milk, add 5 mL of methanol-water mixture (volume ratio 3:2), vortex for 10 min, filter through glass fiber filter paper, purify the filtrate through an AFM1 immunoaffinity column (Zhongjian Weikang product, product number: IAC102), and detect the AFM1 content using the high performance liquid chromatography analysis method described in Example 3.

[0044] Table 1 Aflatoxin Oxidase Bl Effects of CotA and its mutant E188R-S210G-T418A on the removal of AFM1 from raw milk The results are shown in Table 1. Aflatoxin oxidase was used. Bl CotA treatment of raw milk reduced AFM1 residues to 0.42 ng / mL, 0.23 ng / mL, and 0.03 ng / mL after 1 h, 2 h, and 4 h, respectively. Treatment of raw milk with the mutant E188R-S210G-T418A reduced AFM1 residues to 0.31 ng / mL, 0.12 ng / mL, and 0.01 ng / mL after 1 h, 2 h, and 4 h, respectively. The mutant E188R-S210G-T418A showed better results than the maternal parent. Bl CotA.

[0045] Application Example 2: Aflatoxin Oxidase Bl CotA and its mutant E188R-S210G-T418A were used to remove aflatoxin from corn slurry. Add 100 mL of aflatoxin-contaminated corn steep liquor to a 500 mL Erlenmeyer flask, and add 100 mg of the aflatoxin oxidase prepared in Example 3. Bl The aflatoxin residue in corn steep liquor was determined after reacting with CotA or the mutant E188R-S210G-T418A enzyme protein at 37℃ for 6 h.

[0046] The determination method is as follows: Take 10 mL of corn steep liquor, add 5 mL of methanol-water mixture (volume ratio 3:2), vortex for 10 min, filter through glass fiber filter paper, purify the filtrate through a total aflatoxin immunoaffinity column (Zhongjian Weikang product, product number: IAC103), and use the high performance liquid chromatography analysis method described in Example 3 to detect the contents of AFB1, aflatoxin B2 (AFB2), aflatoxin G1 (AFG1), and aflatoxin G2 (AFG2).

[0047] Table 2 Aflatoxin Oxidase Bl The effect of CotA and its mutant E188R-S210G-T418A on the removal of aflatoxin from corn slurry The results are shown in Table 2. Before treatment, the contents of AFB1, AFB2, AFG1, and AFG2 in corn steep liquor were 213.1 ng / mL, 21.5 ng / mL, 46.7 ng / mL, and 12.4 ng / mL, respectively. Aflatoxin oxidase was used to treat the corn steep liquor. Bl After 6 h of CotA treatment, the contents of AFB1, AFB2, AFG1, and AFG2 in corn steep liquor decreased to 63.7 ng / mL, 5.1 ng / mL, 4.6 ng / mL, and 2.4 ng / mL, respectively; aflatoxin oxidase... Bl CotA exhibited degradation rates of 70.11%, 76.28%, 90.15%, and 80.65% for AFB1, AFB2, AFG1, and AFG2, respectively. After treatment with the mutant E188R-S210G-T418A for 6 h, the contents of AFB1, AFB2, AFG1, and AFG2 in corn steep liquor decreased to 13.4 ng / mL, 1.3 ng / mL, 1.5 ng / mL, and 0.3 ng / mL, respectively; the degradation rates of AFB1, AFB2, AFG1, and AFG2 by the mutant E188R-S210G-T418A were 93.71%, 93.95%, 96.79%, and 97.58%, respectively. The mutant E188R-S210G-T418A showed better performance than the parent mutant. Bl CotA.

[0048] 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 within the protection scope of the present invention.

Claims

1. An aflatoxin oxidase mutant E188R-S210G-T418A, characterized in that: The amino acid sequence is shown in SEQ ID NO.

2.

2. The aflatoxin oxidase mutant E188R-S210G-T418A according to claim 1, characterized in that: The mutant E188R-S210G-T418A is an aflatoxin oxidase with the amino acid sequence shown in SEQ ID NO.

1. Bl CotA was obtained by simultaneously mutating amino acids at positions 188, 210, and 418; specifically, glutamic acid at position 188 was mutated to arginine, serine at position 210 was mutated to glycine, and threonine at position 418 was mutated to alanine.

3. The gene encoding the aflatoxin oxidase mutant E188R-S210G-T418A, characterized by: The aflatoxin oxidase mutant E188R-S210G-T418A as described in claim 2 is encoded.

4. The gene encoding the aflatoxin oxidase mutant E188R-S210G-T418A according to claim 3, characterized in that: The nucleotide sequence is shown in SEQ ID NO.

4.

5. A recombinant expression vector containing the encoding gene of claim 4.

6. A recombinant expression strain containing the recombinant expression vector of claim 5.

7. The method for preparing the aflatoxin oxidase mutant E188R-S210G-T418A according to any one of claims 1-2, characterized in that, The steps are as follows: (1) Cultivate the recombinant expression strain according to claim 6 and induce the expression of aflatoxin oxidase mutant E188R-S210G-T418A; (2) The expressed aflatoxin oxidase mutant E188R-S210G-T418A was recovered and purified.

8. The use of the aflatoxin oxidase mutant E188R-S210G-T418A according to any one of claims 1-2 in removing aflatoxin from grains, feed, nuts, milk, corn steep liquor or herbal extracts.

9. The application according to claim 8, characterized in that: The aflatoxin includes at least one of aflatoxin B1, aflatoxin B2, aflatoxin G1, aflatoxin G2, and aflatoxin M1.

10. The application according to claim 9, characterized in that: Add 10-100 mg of aflatoxin oxidase mutant E188R-S210G-T418A to every 100 mL of corn steep liquor or milk.

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