Metarhizium anisopliae IFST-OT3 and its produced detoxification enzyme and its application

Abstract

The present invention provides a strain of brown metarhizium IFST-OT3 and the detoxification enzyme produced by it and its application. The present invention provides a strain of brown metarhizium IFST-OT3 (deposit number CGMCC No. 40349) with strong degradation ability for ochratoxins A and B, as well as the detoxification enzyme (degradation enzyme) Amh1 produced thereby and the nucleotide sequence encoding the amidase, and also provides the optimal physicochemical properties of the enzyme, including the influence of temperature, pH and metal ions. The amidase treats a substrate concentration of 1 μg / ml OTA toxin in a buffer system at 60°C and pH 6.0, and the degradation efficiency is as high as 100% within 3 minutes. Amh1 has strong adaptability to temperature and acidic pH. The strain IFST-OT3 and the detoxification enzyme produced thereby can be used for the degradation of ochratoxins in agricultural products, and has broad application prospects.

Description

Technical Field

[0001] The present invention relates to the field of microbiology and biological detoxification technology, and in particular to a brown Metarhizium IFST-OT3 and a detoxification enzyme produced by the same and its application. Background Art

[0002] Ochratoxin A (OTA) is a secondary metabolite produced by Aspergillus and Penicillium fungi. In 1993, the International Agency for Research on Cancer classified OTA as a Class 2B carcinogen. Furthermore, studies have shown that OTA exhibits nephrotoxic, neurotoxic, and immunotoxic properties (Malir et al., 2016, Toxins 8:191). Ochratoxin B (OTB) is a precursor of OTA, and its molecular structure lacks one chlorine atom. OTA and OTB can contaminate a variety of agricultural products, including corn, wheat, sorghum, fruit, dairy products, and meat products (Wang et al., 2022, Frontiers in Microbiology, 13:857-726), causing serious food safety concerns. The presence of OTA in food and feed can hinder import and export trade, increase medical costs for humans and animals, and result in significant economic losses.

[0003] Taking a variety of methods to remove contaminated OTA from agricultural products is an indispensable way to avoid economic losses. At present, the food and feed industry mainly uses physical, chemical and biological methods for detoxification, among which biological detoxification is safe, efficient and pollution-free, and has the greatest application prospects. Biological detoxification methods can be divided into two categories: one is based on the adsorption of microorganisms themselves, and the other is the degradation of OTA by metabolites produced by microorganisms, such as enzymes. There are a wide range of microbial resources that can degrade OTA in nature, and some bacteria have a degradation rate of OTA of up to 90%. Bacillus amyloliquefaciens achieved a degradation rate of 98.5% for OTA within 24 hours (Chang et al., 2015, Food Additives & Contaminants: Part A, 32, 564-571). Bacillus subtilis, isolated from elk feces, degraded OTA by 97.6% within 24 hours (Shi et al., 2014, Journal of the Science of Food and Agriculture, 94:1879-1885). Alcaligenes faecalis degraded OTA by 97% within 48 hours (Zhang et al., 2017, Journal of Applied Microbiology, 123:661-668).

[0004] Currently, the discovery of biodetoxification enzymes and their encoding genes can achieve precise and efficient OTA removal, improve the stability of detoxification effects, and accumulate key core technologies for biological detoxification methods. Currently, there are few reports on detoxification mechanisms and toxin degradation genes, and the discovery of new OTA biodegradation enzymes is urgently needed. Summary of the Invention

[0005] The purpose of the present invention is to provide a brown green anisopliae Metarhizium brunneum IFST-OT3 and its application in biological detoxification.

[0006] Another object of the present invention is to provide a detoxification enzyme produced by Metarhizium anisopliae IFST-OT3 and its application.

[0007] In order to achieve the purpose of the present invention, in the first aspect, the present invention provides a brown green muscardine Metarhizium brunneum IFST-OT3 isolated and purified from a soil sample. The fungus has been deposited in the General Microbiology Center of the China Culture Collection Administration, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Postal Code 100101, Deposit Number CGMCC No. 40349, and Deposit Date October 28, 2022.

[0008] In a second aspect, the present invention provides a bacterial agent containing the brown Metarhizium IFST-OT3.

[0009] In a third aspect, the present invention provides the use of the brown Metarhizium anisopliae IFST-OT3 or its bacterial agent in the detoxification of ochratoxins A and B.

[0010] In a fourth aspect, the present invention provides a detoxification enzyme Amh1 produced by the brown Metarhizium IFST-OT3, wherein the detoxification enzyme Amh1 comprises or consists of the following amino acid sequence:

[0011] i) the amino acid sequence shown in SEQ ID NO: 1; or

[0012] ii) an amino acid sequence obtained by linking a tag to the N-terminus and / or C-terminus of i); or

[0013] iii) An enzyme having the same function obtained by substituting, deleting and / or adding one or more amino acids in the amino acid sequence of i) or ii).

[0014] In a fifth aspect, the present invention provides a nucleic acid molecule encoding the detoxification enzyme Amh1, the nucleotide sequence of which is shown in SEQ ID NO: 2.

[0015] In a sixth aspect, the present invention provides a biological material containing the nucleic acid molecule, wherein the biological material includes but is not limited to recombinant DNA, an expression cassette, a transposon, a plasmid vector, a viral vector, an engineered bacterium or a transgenic cell line.

[0016] In a seventh aspect, the present invention provides any of the following applications of the detoxification enzyme Amh1:

[0017] 1) Used to degrade ochratoxins A and B;

[0018] 2) Used for preparing detoxification agents for ochratoxins A and B.

[0019] In an eighth aspect, the present invention provides a method for degrading ochratoxin A, comprising contacting the detoxifying enzyme Amh1 with ochratoxin A in a buffer system at 30-80° C. and pH 4.0-8.0.

[0020] Preferably, the concentration of ochratoxin A in the system is 1-5 μg / ml.

[0021] Preferably, the molar ratio of detoxifying enzyme Amh1 to ochratoxin A is 1:130.

[0022] The present invention realizes heterologous high expression of the detoxification enzyme in a prokaryotic expression system (Escherichia coli), and has extremely high degradation efficiency for ochratoxin.

[0023] By means of the above technical solution, the present invention has at least the following advantages and beneficial effects:

[0024] The present invention provides a strain of Metarhizium anisopliae (IFST-OT3) with strong ability to degrade ochratoxins A and B, as well as a detoxifying enzyme (degrading enzyme) produced by the strain and a nucleotide sequence encoding the amidase. The enzyme's optimal physicochemical properties, including temperature and pH, are also provided. The amidase degrades OTA toxins at a substrate concentration of 1 μg / ml within 3 minutes in a buffer system at 60°C and pH 6.0. The strain IFST-OT3 and the detoxifying enzyme produced by the strain can be used to degrade ochratoxins in agricultural products and have broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1Figures 1 and 2 show the HPLC analysis of a crude IFST-OT3 enzyme solution incubated for 24 hours with OTA and OTB standard samples at a substrate concentration of 1 μg / ml, as well as the molecular mass analysis by HPLC-MS. Figures A and B show the HPLC and HPLC-MS analysis of the OTA standard; Figures C and D show the HPLC and HPLC-MS analysis of OTA and the crude IFST-OT3 enzyme solution after a 24-hour incubation; Figures E and F show the HPLC and HPLC-MS analysis of the OTB standard; and Figures G and H show the HPLC and HPLC-MS analysis of OTB and the crude IFST-OT3 enzyme solution after a 24-hour incubation.

[0026] Figure 2 Figure 1 shows the cloning, prokaryotic expression, and purification of the detoxification enzyme Amh1 gene in a preferred embodiment of the present invention. Figure A shows an agarose gel electrophoresis image of the Amh gene cloned from the cDNA of strain IFST-OT3; Figure B shows an SDS-PAGE electrophoresis image of Amh1 expressed in E. coli, with an empty expression vector serving as a control; and Figure C shows an SDS-PAGE electrophoresis image of purified Amh1.

[0027] Figure 3 The degradation efficiency of amidase Amh1 in a preferred embodiment of the present invention is shown in Figure 1. A represents the effect of different pH values ​​on the degradation efficiency; B represents the effect of different temperatures on the degradation efficiency; C represents the effect of different metal ions on the degradation efficiency; and D represents the degradation time of OTA at concentrations of 1.0 μg / ml, 2.5 μg / ml, and 5.0 μg / ml at the optimal pH of 6.0 and the optimal temperature of 60°C. DETAILED DESCRIPTION

[0028] The following examples are provided to illustrate the present invention but are not intended to limit the scope of the invention. Unless otherwise specified, the examples were performed according to conventional experimental conditions, such as those in Sambrook et al. (Sambrook J & Russell DW, Molecular Cloning: a Laboratory Manual, 2001), or according to the conditions recommended by the manufacturer's instructions.

[0029] Example 1 Isolation and Identification of Metarhizium anisopliae IFST-OT3

[0030] Isolation and purification of brown green anisopliae IFST-OT3: IFST-OT3 was isolated from Beijing farmland soil samples by the following method: 5 g of soil was added to 5 mL of sterile water, shaken on a shaker for 30 min (180 rpm), gradiently diluted 10, 100, and 1000 times, respectively, and spread onto PDA culture medium, inverted and cultured at 28°C for 72 h, and the grown colonies were purified by the plate streak method, and the obtained single colonies were inoculated onto PDA plates for culture.

[0031] On PDA medium, strain IFST-OT3 colonies are fuzzy or flocculent, initially white and dark green after sporulation, with regular, smooth colony margins. The hyphae are septate, smooth, and transparent, with 2-3 phialides on conidiophores. Conidia are unicellular. Strain IFST-OT3 was cultured in PDB medium with shaking. DNA was extracted after 3 days, and EF-1a was amplified (primer sequence: 5'-GTACCTCCCAGGCTGACTGC-3';

[0032] 5'-GTTCTTGGAGTCACCAGCAACG-3'), RPB2

[0033] (5'-GAGGAGCTGAAGAGGACCGG-3'; 5'-AATCGGTGTGTTGGTTCGTCG-3'), beta-tub (5'-CCGTCCATCAGCTCGTTGAG-3';

[0034] The three genes were identified and sequenced. The NCBI accession numbers for these three genes are OP947525, OP947524, and OP947526, respectively. BLAST analysis revealed that all three genes shared the highest sequence identity with homologous genes in Metarhizium brunneum ARSEF 3297.

[0035] Based on the sequencing results and the above microbiological characteristics, strain IFST-OT3 was identified as Metarhizium brunneum, with the deposit number CGMCC No.40349.

[0036] Example 2 Degradation ability of Metarhizium anisopliae IFST-OT3 on OTA and OTB

[0037] At 28°C, Metarhizium anisopliae IFST-OT3 was cultured in PDB liquid medium at 180 rpm for 7 days. The collected mycelium was ground into powder in a mortar with the continuous addition of liquid nitrogen. 1 g of the powder was added to 20 ml of Lysis Buffer (1 mM PMSF, 1% anisole hydrochloride, 10 mM Tris-HCl (pH 7), 150 mM NaCl, 0.5 mM EDTA, 0.01% Triton X-100, 1 mM DTT) and vortexed to mix. The supernatant was obtained by centrifugation at 8000 rpm for 15 minutes at 4°C, and the crude enzyme solution was obtained. 1 ml of the crude enzyme solution was aspirated, and OTA or OTB standard (final concentration of 1 μg / ml) was added. The reaction was incubated at 28°C for 24 hours. 1 ml of methanol was added to terminate the reaction, and the product was detected by high-performance liquid chromatography (HPLC) and high-performance liquid chromatography tandem mass spectrometry (HPLC-MS).

[0038] The results are as follows Figure 1 As shown in (A~H), the peak time of OTA standard is 15.1min( Figure 1 A), whose molecular weight was determined to be 403 ([M+H] + =404, Figure 1 B); After OTA and IFST-OT3 were incubated for 24 h, the peak of OTA disappeared and a new peak appeared at 6.2 min ( Figure 1 C), whose molecular weight is 256 ([MH] + =255, Figure 1 D), the peak time and molecular weight are consistent with the degradation product OTα after the amide bond of OTA is broken; the peak time of OTB standard is 8.9min( Figure 1 E), whose molecular weight was determined to be 369 ([M+H] + =370, Figure 1 F); After incubation of OTB and IFST-OT3 for 24 h, the OTB peak disappeared and a new peak appeared at 4.6 min ( Figure 1 G), whose molecular weight is 222([MH] + =221, Figure 1 The peak time and molecular weight are consistent with those of the degradation product OTβ after amide bond cleavage of OTB. These results indicate that M. anisopliae IFST-OT3 can degrade OTA and OTB into OTα and OTβ.

[0039] Example 3 Construction of expression vector

[0040] The present invention extracts crude fungal protein and measures degradation activity to find that brown Metarhizium anisopliae IFST-OT3 has the ability to degrade OTA into OTα. Through protein separation activity assay, mass spectrometry analysis, gene ontology analysis and protein recombinant expression, an amidase (detoxification enzyme) with degradation activity is finally obtained. The gene Amh1 (GenBank: OP947527) is amplified from the reverse transcribed cDNA of Metarhizium brunneum strain ( Figure 2 , A), when designing primers, a BamHI restriction site was introduced into the upstream primer and a NotI restriction site was introduced into the downstream primer. GGATCC ATGACTAGACGGGTGATTCCCC-3'; downstream primer 5'- GCGGCCGC TCACAACTCCTCTCCCCAAGG-3', the restriction endonuclease sequence is underlined, and a high-fidelity DNA polymerase is used to amplify the product with blunt ends. The target fragment is recovered by gel recovery kit and mixed with 1 μl vector at a ratio of 40 ng. The recombinant plasmid was constructed by ligating the recombinant vector with the recombinant vector and performing blue-white screening and sequencing to obtain the correct gene fragment. The pET28a(+) vector was linearized with BamHI and NotI restriction endonucleases, and the digested vector and fragment were ligated using a DNA Ligation Kit (MightyMix, Takara, Japan) at 16°C for 3 hours. The recombinant plasmid was transformed into E. coli DH5α, and the correct expression recombinant plasmid was verified by restriction endonuclease sequencing. The plasmid was finally transferred into the expression strain E. coli BL21(DE3).

[0041] Example 4 Heterologous expression of amidase (detoxification enzyme) gene

[0042] A single clone of the transformed E. coli BL21 (DE3) strain from Example 2 was cultured overnight in 10 mL of LB liquid medium containing 80 μg / mL kanamycin. The culture was inoculated into 1 L of LB liquid medium (containing kanamycin) at a 1% inoculum size and cultured at 37°C and 180 rpm. The bacterial concentration (OD) was determined. 600 , when the OD value is 0.6-0.8, add 1mg / ml IPTG solution at a ratio of 1 / 2000 (the final concentration of IPTG is 1mM). After adding, adjust the shaker temperature to 16℃. When the bacterial solution cools to 16℃, shake and induce at 100rpm for about 16 hours. Centrifuge 1L of bacterial solution at 4℃, 4000rpm for 10 minutes, discard the supernatant, dissolve the bacteria in the lysate and stir for 15-20 minutes (30mL W1 buffer: 50mM Tris-HCl pH 8.0, 300mMNaCl; 1mM PMSF; 10mM MgCl2; 20μg / mL Lysozyme; 1μg / mL DNase I). Homogenize at 4℃ high pressure (pressure 1000MPa) for 10-15 minutes (depending on the amount of bacteria) until the bacterial solution is clear. Centrifuge the clarified bacterial solution at 4℃, 12000rpm for 45 minutes, and retain the supernatant as the crude enzyme solution. Figure 2 As shown in B, the SDS-PAGE electrophoresis of BL21 expressing Amh1 shows an additional band between 55 kDa and 70 kDa compared with BL21 expressing an empty vector, which is presumably the protein expressed by the Amh1 gene.

[0043] Example 5 Protein Purification

[0044] A chromatography column containing approximately 1.5 mL of Ni filler was pretreated by rinsing with Ni-Elute buffer (50 mM Tris-HCl, pH 8.0, 300 mM imidazole) for two column volumes and ddH2O for two column volumes. One column volume of W1 buffer, as described in Example 3, was then rinsed. The retained supernatant from Example 3 was then loaded onto the chromatography column. To remove nonspecifically bound contaminants, one column volume of W1 buffer was added to the chromatography column after the sample was drained. After the W1 buffer was drained, one column volume of W2 buffer (50 mM Tris-HCl, pH 8.0, 500 mM NaCl, 20 mM imidazole) was added. To remove high salt content from the purified protein, one column volume of W3 buffer (50 mM Tris-HCl, pH 8.0) was added after the W2 buffer was drained. Finally, Ni-Elute buffer was added to the chromatography column to elute the target protein. The effluent was collected and assayed using a Bradford assay until the blue color became lighter.

[0045] The purified protein collected in the previous step is concentrated by anion exchange chromatography column, and then ultrafiltration is performed by selecting a suitable ultrafiltration tube according to the molecular weight of the protein (using a 30KDa ultrafiltration tube). After ultrafiltration concentration, the purified protein is concentrated again by gel filtration chromatography protein purification method (molecular sieve) and the concentration is measured. The purified protein results are detected by SDS-PAGE electrophoresis, as shown in FIG. Figure 2 As shown in Figure C, the apparent molecular weight of the Amh1 protein is consistent with the theoretical molecular weight (55.4 KDa). The purified protein was quickly frozen with liquid nitrogen and stored in a -80°C freezer for future use.

[0046] Example 6 Enzyme activity determination

[0047] The purified protein Amh1 in Example 5 was diluted to 2.5 μg / mL for enzyme activity determination (the dilution buffer was Tris-HCl pH 6.0). The reaction system was 1 mL, and the purified protein Amh1 at a concentration of 2.5 μg / mL was mixed with 1 μL of 1000 μg / ml OTA toxin standard sample. The pH gradient was set to 3, 4, 5, 6, 7, 8, 9, 10, and the degradation experiment was carried out at 28°C. The results showed that the enzyme had a high efficiency in degrading OTA in a slightly acidic environment of pH 4-7. After incubation for 40 minutes, OTA could be completely degraded under the conditions of pH 5-7 ( Figure 3, A). The temperature gradient was set at 20℃, 30℃, 40℃, 50℃, 60℃, 70℃, and 80℃, and the degradation experiment was carried out under pH 6. After incubation for 40 minutes, OTA was completely degraded at 30-70℃; after incubation for 20 minutes, OTA was completely degraded at 40-70℃; after incubation for 10 minutes, OTA was completely degraded at 60℃ ( Figure 3 , B). The above results show that the enzyme can efficiently degrade OTA at pH 4-7 and temperature 30-70℃, and exhibits good acid and heat resistance.

[0048] To further study the effects of nine metal ions on the activity of Amh1, ZnSO4, MnSO4, FeSO4, Fe2(SO4)3, NiSO4, CuSO4, MgSO4, LiCl2, and CaCl2 were added to the reaction system to make the final concentration of metal ions 50 mM, and the reaction was carried out at 28°C and pH 6 for 20 minutes. 2+ 、Zn 2+ 、Mn 2+ , Ca 2+ Significantly improve the degradation activity of Ni 2+ The enzyme activity is reduced, while in Fe 2+ 、Fe 3+ 、Cu 2+ Under the presence of , Amh1 cannot degrade OTA ( Figure 3 , C). The degradation rate of OTA at 1 μg / ml by Amh1 was tested under the optimum temperature (60°C) and pH 6.0. The results showed that when the concentration of Amh1 was 1 μg / ml, OTA was completely degraded within 11 minutes; when the concentration was 2.5 μg / ml, OTA was completely degraded within 5 minutes; when the concentration was 5 μg / ml, OTA was completely degraded within 3 minutes ( Figure 3 , D). Compared with other degradation enzymes, Amh1 has a very high degradation efficiency.

[0049] Although the present invention has been described in detail above using general descriptions and specific embodiments, it will be apparent to those skilled in the art that modifications and improvements may be made based on the present invention. Therefore, such modifications and improvements, which do not depart from the spirit of the present invention, are intended to be within the scope of protection claimed herein.

Claims

1. Brown Metarhizium ( Metarhizium brunneum )IFST-OT3, characterized in that, The deposit number is CGMCC No.40349.

2. A bacterial agent containing the brown Metarhizium IFST-OT3 according to claim 1.

3. Use of the brown Metarhizium anisopliae IFST-OT3 or its bacterial agent according to claim 1 in the detoxification of ochratoxin A and ochratoxin B.

4. The detoxification enzyme Amh1 produced by the brown Metarhizium IFST-OT3 according to claim 1, characterized in that The amino acid sequence of the detoxification enzyme Amh1 is shown in SEQ ID NO:

1.

5. A nucleic acid molecule encoding the detoxification enzyme Amh1 according to claim 4.

6. A recombinant DNA, expression cassette, transposon, plasmid vector, viral vector, engineered bacteria or transgenic cell line containing the nucleic acid molecule of claim 5.

7. Any of the following uses of the detoxification enzyme according to claim 4: 1) Used to degrade ochratoxin A and ochratoxin B; 2) Used for preparing detoxification agents for ochratoxin A and ochratoxin B.

8. A method for degrading ochratoxin A, characterized in that: The detoxification enzyme Amh1 according to claim 4 is contacted with ochratoxin A in a buffer system at 30-80° C. and pH 4.0-8.

0.

9. The method according to claim 8, characterized in that The concentration of ochratoxin A in the system is 1-5 .

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