Aflatoxin B1 degrading enzyme as well as preparation method and application thereof

Abstract

The invention provides an aflatoxin B1 degrading enzyme as well as a preparation method and application thereof, and belongs to the technical field of agricultural biology, and a nucleotide sequence of the aflatoxin B1 degrading enzyme is shown as SEQ ID NO.1. The invention provides an aflatoxin B1 degrading enzyme as well as a preparation method and application thereof. The purpose of efficiently degrading aflatoxin B1 by using a novel degrading enzyme is achieved.

Description

Technical Field

[0001] The invention relates to the field of agricultural biotechnology, and in particular to an aflatoxin B1 degrading enzyme and a preparation method and application thereof. Background Art

[0002] With the growth of global population and economic development, food security issues are increasingly attracting the attention of governments and international organizations. However, the contamination of fungi in food production has become an important factor restricting food security. According to the Food and Agriculture Organization of the United Nations (FAO), about 25% of the world's food is contaminated with fungi every year, causing economic losses of up to tens of billions of dollars. At the same time, in order to fully implement my country's food security strategy and ensure stable and sustained economic growth, it is urgent to control the contamination of fungi in food. The fungal toxins that contaminate food mainly include aflatoxins, zearalenone, deoxynivalenol and ochratoxin. Among them, aflatoxin B1 (AFB1) is the most serious, the most toxic and carcinogenic. It was listed as a Class I carcinogen by the International Agency for Research on Cancer (IARC) in 1993.

[0003] At present, more than 100 countries and regions have established maximum limit standards for the content of AFB1 in food and feed. China's standard for the limit of mycotoxins in food - GB 2761-2017 "National Food Safety Standard Limit of Mycotoxins in Food" includes 6 major categories and 16 minor categories of food, and the limit range of AFB1 is 0.5μg / kg~20μg / kg. In 2006, the EU stipulated that the maximum limit value of aflatoxin is between 0.1μg / kg and 12μg / kg. For the limit standard of AFB1 in feed in China, please refer to GB13078-2017 "Feed Hygiene Standard", which sets AFB1 limit requirements for 2 major categories and 13 minor categories of feed raw materials and feed products, with the maximum limit content between 10μg / kg and 50μg / kg.

[0004] Biotechnology has the advantages of being green, efficient, specific, and free of secondary pollution, and has gradually become a research hotspot for removing AFB1 contamination from grains. Bioenzymatic degradation of AFB1 is of great significance in reducing grain losses caused by fungal toxin contamination and ensuring the safety of grain quantity and quality.

[0005] The invention patent with publication number CN118256459A and titled A recombinant enzyme and its application in degrading aflatoxin B1 discloses a recombinant enzyme that can degrade AFB1. However, the degradation rate of AFB1 in the above technical solution can only reach about 60% under optimal conditions. Summary of the invention

[0006] In view of this, the present invention provides an aflatoxin B1 degrading enzyme and a preparation method and application thereof, so as to achieve the purpose of efficiently degrading aflatoxin B1 using the degrading enzyme.

[0007] To achieve the above object, the present invention provides an aflatoxin B1 degrading enzyme, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0008] Optionally, its amino acid sequence is shown in SEQ ID NO.2.

[0009] Optionally, the aflatoxin B1 degrading enzyme is used at a pH of 7-10 and a temperature of 20-60°C.

[0010] In order to achieve the above object, the present invention also provides a method for preparing an aflatoxin B1 degrading enzyme, comprising the following steps: S1, synthesize the gene encoding aflatoxin B1 degrading enzyme and connect it between the BamHI and XhoI restriction sites of pET-21a; S2, transform E. coli competent cells BL21 (DE3), select positive transformants through Amp resistance plate screening, extract transformation plasmids, and perform double enzyme digestion verification and sequencing to confirm that the correct recombinant strain is obtained; S3, inoculating the recombinant strain into LB culture medium to obtain a bacterial solution, transferring the bacterial solution into LB culture medium for culture, adding IPTG for induction, and obtaining a fermentation broth; S4. Resuspend the bacterial cells obtained by centrifugation of the fermentation broth, centrifuge again, wash the bacterial cells, resuspend them again, centrifuge after ultrasonication, collect the supernatant, and purify it using a nickel ion affinity chromatography column to obtain aflatoxin B1 degrading enzyme.

[0011] Optionally, the conditions for culturing the recombinant strain in S3 in LB culture medium are a temperature of 37°C, a rotation speed of 180 rpm, and a time of 11 to 13 hours; the conditions for culturing the bacterial solution in LB culture medium are a temperature of 37°C, a rotation speed of 180 rpm, and a time of 2 to 3 hours.

[0012] Optionally, when the bacterial solution is transferred to LB medium, the bacterial solution is 1% of the weight of the LB medium; the final concentration of IPTG is 1 mmol / L, and the induction time is 15-17 hours. Optionally, the centrifugation temperature in S4 is 4°C, the rotation speed is 12000 rpm, and the time is 10-30 min.

[0013] Alternatively, resuspend in S4 using pH 7.0 phosphate buffer.

[0014] To achieve the above object, the present invention also provides the application of aflatoxin B1 degrading enzyme in feed additives.

[0015] Optionally, the feed additive is a liquid preparation.

[0016] The above technical solution of the present invention includes at least the following beneficial effects: The aflatoxin B1 degrading enzyme provided by the present invention enriches the types of aflatoxin B1 degrading enzymes, the aflatoxin B1 degradation rate can be as high as more than 90%, and aflatoxin B1 can be degraded efficiently, thereby improving the application value of the aflatoxin B1 degrading enzyme. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is a purification detection diagram of the target protein in Example 1 of the present invention; Figure 2 High performance liquid chromatogram of aflatoxin B1 degrading enzyme degrading aflatoxin B1 in Example 2 of the present invention; Figure 3 is a high performance liquid chromatogram of aflatoxin B1 degrading enzyme degrading aflatoxin B1 in Example 7 of the present invention; Figure 4 This is a graph showing the degradation rates of aflatoxin B1 degrading enzymes of Examples 2 to 5 of the present invention; Figure 5 It is the degradation rate diagram of aflatoxin B1 degrading enzyme at different pH; Figure 6 This is a graph showing the degradation rate of aflatoxin B1 degrading enzyme at different degradation times. DETAILED DESCRIPTION

[0018] To make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the following will be combined with the appended drawings of the embodiments of the present invention. Figure 1-6 , the technical scheme of the embodiment of the present invention is clearly and completely described. Obviously, the described embodiment is a part of the embodiment of the present invention, not all of the embodiments. Based on the described embodiment of the present invention, all other embodiments obtained by ordinary technicians in this field belong to the scope of protection of the present invention.

[0019] The biological materials and experimental reagents involved in the embodiments of the present invention are as follows: Vector E. coli expression vector pET-21a was purchased from Invitrogen.

[0020] Bacterial strain: BL21 (DE3) chemically competent cells, purchased from Invitrogen.

[0021] Experimental reagents: Aflatoxin B1 was purchased from Anolon; other reagents were domestic analytical grade.

[0022] Example 1 Preparation of aflatoxin B1 degrading enzyme The aflatoxin B1 degrading enzyme encoding gene was synthesized by Anhui General Biosystem Co., Ltd. and ligated between the BamHI and XhoI restriction sites of pET-21a. Its nucleotide sequence is shown in SEQ ID NO. 1. The aflatoxin B1 degrading enzyme encoding gene was codon optimized to make it more conducive to expression in Escherichia coli.

[0023] The competent E. coli cells BL21 (DE3) were transformed, and the positive transformants were selected by Amp resistance plate screening. The transformation plasmid was extracted, and double enzyme digestion verification and sequencing were performed to determine that the correct recombinant strain was obtained, and its amino acid sequence is shown in SEQ ID NO.2.

[0024] The above recombinant strain was inoculated into 50 mL LB culture medium, and cultured at 37°C, 180 rpm with shaking for 12 h. Then, it was transferred to 400 mL LB medium at a 1% ratio, and cultured at 37°C, 180 rpm with shaking for about 3 h (OD600≈0.6). A final concentration of 1 mM IPTG was added, and the culture was induced at 16°C for 16 h.

[0025] Collect the fermentation broth, centrifuge at 4°C, 12000rpm for 30min, discard the supernatant; resuspend the bacteria in pH 7.0 phosphate buffer, centrifuge at 4°C, 12000rpm for 30min, discard the supernatant, and repeat washing the bacteria three times. Then resuspend the bacterial cells in phosphate buffer (20mM, pH 7.0), ultrasonically disrupt, centrifuge at 4°C, 12000rpm for 10min, collect the supernatant and filter. Since the C-terminus of the expressed aflatoxin B1 degrading enzyme carries a histidine tag (6×His), a nickel ion affinity chromatography column (Ni2+-NTA) is used to purify the recombinant protein. For the steps of equilibration, loading, elution, etc., please refer to the Qiagen user manual. The purified protein is ultrafiltered with a cutoff tube (10kDa) to remove the imidazole contained therein. The purification results of the target protein can be detected by SDS-PAGE electrophoresis, such as Figure 1 .

[0026] Example 2 Detection of the degradation activity of aflatoxin B1 by aflatoxin B1 degrading enzyme Aflatoxin B1 was dissolved in methanol to prepare a 100 μg / mL mother solution, and the experiment was carried out according to the following 1000 μL reaction system: 980 μL glycine-NaOH buffer (0.1M, pH 9.0), 10 μL aflatoxin B1 degrading enzyme (final enzyme activity 1U / mL), 10 μL aflatoxin B1 solution mother solution. The chromatographic conditions for the HPLC detection of AFB1 were as follows: chromatographic column: Waters-Acchrom Tnature C18 (4.6×150mm, 5μm); mobile phase: methanol-water (50:50); flow rate: 1mL / min; pump pressure: 162bar; injection volume: 10μL; fluorescence detector detection wavelength: λex=360nm, λem=440nm; acquisition time: 10min.

[0027] The system without aflatoxin B1 degrading enzyme was used as a control. After the reaction was carried out at 30°C for 4 hours, 3 mL of methanol was added to terminate the reaction. High performance liquid chromatography was used to detect whether the aflatoxin B1 degrading enzyme had degradation activity on aflatoxin B1 based on the change in AFB1 concentration. Figure 2 .

[0028] like Figure 2 As shown, the control group sample had a strong absorption peak at retention time RT = 8.246min, while a smaller AFB1 target peak was detected in the group with aflatoxin B1 degrading enzyme. According to the calculation of the absorption peak area, 98% of the AFB1 molecules had been degraded, indicating that the recombinant aflatoxin B1 degrading enzyme has the activity of degrading aflatoxin B1 and the degradation rate is high.

[0029] Example 3 Aflatoxin B1 was dissolved in methanol to prepare a 100 μg / mL mother solution, and the experiment was carried out according to the following 1000 μL reaction system: 980 μL glycine-NaOH buffer (0.1M, pH 9.0), 10 μL aflatoxin B1 degrading enzyme (final enzyme activity 1U / mL), 10 μL aflatoxin B1 solution. The chromatographic conditions for the HPLC detection of AFB1 were as follows: chromatographic column: Waters-AcchromTnature C18 (4.6×150mm, 5μm); mobile phase: methanol-water (50:50); flow rate: 1mL / min; pump pressure: 162bar; injection volume: 10μL; fluorescence detector detection wavelength: λex=360nm, λem=440nm; acquisition time: 10min.

[0030] The system without aflatoxin B1 degrading enzyme was used as a control. After the reaction was carried out at 20°C for 4 hours, 3 mL of methanol was added to terminate the reaction. The degradation rate of aflatoxin B1 by aflatoxin B1 degrading enzyme was analyzed based on the change in AFB1 concentration using high performance liquid chromatography.

[0031] Example 4 Compared with Example 2, the only difference is that after the reaction is carried out at 40°C for 4 hours, 3 mL of methanol is added to terminate the reaction, and high performance liquid chromatography is used to analyze the degradation rate of aflatoxin B1 by aflatoxin B1 degrading enzyme based on the change in AFB1 concentration. The rest is consistent with Example 2.

[0032] Example 5 Compared with Example 2, the only difference is that after the reaction is carried out at 60°C for 4 hours, 3 mL of methanol is added to terminate the reaction, and high performance liquid chromatography is used to analyze the degradation rate of aflatoxin B1 by aflatoxin B1 degrading enzyme based on the change in AFB1 concentration. The rest is consistent with Example 2.

[0033] Example 6 The feed additive containing aflatoxin B1 degrading enzyme is a liquid preparation, and the aflatoxin B1 degrading enzyme is dissolved in glycine-NaOH buffer (0.1M, pH 9.0) at a concentration of 10U / mL to obtain the additive, which is stored at 4°C or room temperature.

[0034] Example 7 Naturally moldy peanut meal was put into tissue culture bottles, 5g of peanut meal in each bottle, and appropriate amount of water was added to fully dissolve the peanut meal. 1mL of additive was added as the treatment group, and 1mL of sterile water was added to the control group. The mixture was shaken well and cultured at a constant temperature of 30°C for 4h. Aflatoxin B1 in corn was detected by HPLC.

[0035] The chromatographic conditions for the detection of AFB1 by HPLC were as follows: chromatographic column: Waters-Acchrom Tnature C18 (4.6×150mm, 5μm); mobile phase: methanol-water (50:50); flow rate: 1mL / min; pump pressure: 162bar; injection volume: 10μL; fluorescence detector detection wavelength: λex=360nm, λem=440nm; acquisition time: 10min.

[0036] The degradation rate of AFB1 was determined to be 99.28%, as shown in the HPLC test chart. Figure 3 .

[0037] The aflatoxin B1 degrading enzymes prepared in Examples 2 to 5 were subjected to degradation experiments to obtain degradation rates. Figure 4 .Depend on Figure 4 It can be seen that the only difference between Examples 2, 3, 4 and 5 is the reaction temperature, and the degradation rates of the aflatoxin B1 degrading enzymes prepared in Examples 2 to 5 are all above 90%.

[0038] Test Example 1 Based on Example 2, the pH in the reaction system was changed to test the effect of pH on the activity of aflatoxin B1 degrading enzyme in degrading aflatoxin B1.

[0039] The pH in the reaction system was 980 μL buffer (0.1 M glycine-HCl, pH 2; 0.1 M Na2HPO4-sodium citrate buffer, pH 3-7; 0.1 M Tris-HCl buffer, pH 8-9; 0.1 M glycine-NaOH buffer, pH 10), 10 μL aflatoxin B1 degrading enzyme (final enzyme activity 1 U / mL), and 10 μL aflatoxin B1 solution.

[0040] The reaction was carried out at 30°C for 4 hours, and then 3 mL of methanol was added to terminate the reaction. The residual aflatoxin B1 content in the system was detected by the method in Example 2. Figure 5 shown.

[0041] Depend on Figure 5 It can be seen that under alkaline conditions, the activity of aflatoxin B1 degrading enzyme in degrading aflatoxin B1 is relatively high, and the optimal pH is 9.0.

[0042] Test Example 2 Based on Example 2, the reaction time in the reaction system was changed to test the effect of the reaction time on the activity of aflatoxin B1 degrading enzyme in degrading aflatoxin B1.

[0043] The reaction system consisted of 980 μL glycine-NaOH buffer (0.1 M, pH 9.0), 10 μL aflatoxin B1 degrading enzyme (final enzyme activity 1 U / mL), and 10 μL aflatoxin B1 solution.

[0044] After the reaction was carried out for 1, 2, 4, 8, 12, and 24 hours, 3 mL of methanol was added to terminate the reaction. The residual aflatoxin B1 content in the system was detected by the method in Example 2. The results are shown in FIG. Figure 6 shown.

[0045] Depend on Figure 6 It can be seen that when other conditions are the same, the activity of aflatoxin B1 degrading enzyme in degrading aflatoxin B1 increases with time, but the degradation rate changes little after 4 hours. Therefore, 4 hours is a suitable degradation time.

[0046] The SEQ ID NO.1 of this application is as follows: <110> Henan Yiwan Zhongyuan Biotechnology Co., Ltd. <120> Bacterial laccase Lac_P.E., encoding gene and its application and feed additive <160> 1 <170> PatentIn version 3.5 <211> 1539 <212> DNA <213> Lentibacillus sp. <221> multicopper oxidase gene <400> 1 The SEQ ID NO.2 of the present application is as follows: <211> 513 <212> PRT <213> Lentibacillus sp. <221> Multicopper oxidase <400> 2 mnkkltkfvd alpipkilkp lrkdkneayy evemtefrhk mhrdlrptrl wgyngqfpgp 61 iidvsrgkpi qvkwenklpd khflpidksf hdldelpevr tvthlhgset kpesdgypea 121 wytrdfretg pefktdvyhy pnhqhgatlw yhdhamgitr lniyaglagm yiirderekh 181 mklpsgdyei pliimdrsfh dngdlfypsq pddtqenwpn psirpffnge tnvvngkiwp 241 yleveprkyr frilnaantr ayqlhlgsgq sfyqigsdgg lmrktakldn laiepaerfd 301 viidfserkg etitlkndlg pnadpedetd dvmqfkvnlp lstedrsrip rnltripslr 361 qnnikhirnl kligstdnlg rpllllnnkk wgdpvtetpe lgstevwsft nvtnfthpih 421 ihliqfqvld rrsfdldryn kdgeivftgt aeppksners wkdtvaapag qitrvivqfg 481 pftgdyvwhc hilehedydm mrpmrviepg kws The above are preferred embodiments 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 aflatoxin B1 degrading enzyme, characterized in that: Its nucleotide sequence is shown in SEQ ID NO.

1.

2. The aflatoxin B1 degrading enzyme according to claim 1, characterized in that Its amino acid sequence is shown in SEQ ID NO.

2.

3. The aflatoxin B1 degrading enzyme according to claim 1, characterized in that The pH of the aflatoxin B1 degrading enzyme when degrading aflatoxin B1 is 7-10 and the temperature is 20-60°C.

4. A method for preparing an aflatoxin B1 degrading enzyme as claimed in claim 1, characterized in that: The following steps are involved: S1, synthesize the gene encoding aflatoxin B1 degrading enzyme and connect it between the BamHI and XhoI restriction sites of pET-21a; S2, transform E. coli competent cells BL21 (DE3), select positive transformants through Amp resistance plate screening, extract transformation plasmids, and perform double enzyme digestion verification and sequencing to confirm that the correct recombinant strain is obtained; S3, inoculating the recombinant strain into LB culture medium to obtain a bacterial solution, transferring the bacterial solution into LB culture medium for culture, adding IPTG for induction, and obtaining a fermentation broth; S4. Resuspend the bacterial cells obtained by centrifugation of the fermentation broth, centrifuge again, wash the bacterial cells, resuspend them again, centrifuge after ultrasonication, collect the supernatant, and purify it using a nickel ion affinity chromatography column to obtain aflatoxin B1 degrading enzyme.

5. The method for preparing aflatoxin B1 degrading enzyme according to claim 1, characterized in that: The conditions for culturing the recombinant strain in S3 in LB culture medium are 37°C, 180 rpm, and 11-13 h; the conditions for culturing the bacterial solution in LB culture medium are 37°C, 180 rpm, and 2-3 h.

6. The method for preparing aflatoxin B1 degrading enzyme according to claim 1, characterized in that: When the bacterial solution is transferred to the LB medium, the bacterial solution is 1% of the weight of the LB medium; the final concentration of the IPTG is 1 mmol / L, and the induction time is 15-17 hours.

7. The method for preparing aflatoxin B1 degrading enzyme according to claim 1, characterized in that: During centrifugation in S4, the temperature is 4°C, the speed is 12000 rpm, and the time is 10-30 min.

8. The method for preparing aflatoxin B1 degrading enzyme according to claim 1, characterized in that: Resuspend in S4 using pH 7.0 phosphate buffer.

9. Use of the aflatoxin B1 degrading enzyme according to any one of claims 1 to 8 as a feed additive.

10. The aflatoxin B1 degrading enzyme according to claim 9 is used as a feed additive, characterized in that: The feed additive is a liquid preparation.

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