Brevibacillus for degrading OTA, OTA degrading enzyme and its application

Abstract

The present invention discloses a Brevibacillus strain for degrading OTA, an OTA degrading enzyme, and their applications. The present invention first discloses the Brevibacillus strain and its application in degrading OTA. The present invention further discloses the OTA degrading enzyme and its application in degrading OTA. The Brevibacillus strain ASAG55 of the present invention and two OTA degrading enzymes mined from the Brevibacillus strain ASAG55 have the functions of efficiently degrading OTA and biological control of OTA, and have good application prospects in the fields of OTA degradation and prevention and control.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology. More specifically, it relates to Brevibacillus brevis for degrading OTA, OTA degrading enzyme and its application. Background Art

[0002] Ochratoxin A (OTA) is a widely distributed mycotoxin, mainly produced by strains such as Aspergillus niger, Aspergillus ochraceus, and Aspergillus carbonarius. Research shows that OTA has strong hepatotoxicity and nephrotoxicity, and has teratogenic, mutagenic and carcinogenic effects. OTA is also considered to be one of the main pathogenic factors of Balkan endemic nephropathy. OTA widely exists in products such as grains and grain products, spices and coffee beans, seriously endangering human health. Many countries have established limited standards for OTA. Currently, more than 40 countries and regions in the world have stipulated the limits of OTA in grains and their products, fruit wines, dried fruits and infant foods. Due to the pollution and toxicity of OTA, OTA has also caused huge economic losses.

[0003] The degradation and removal of OTA include biological, physical and chemical methods. Physical methods will produce secondary pollution, have low detoxification efficiency and cause losses to the nutrition of food; chemical methods are also not specific and will bring secondary pollution; biological methods have the advantages of high efficiency, specificity and greenness, and are widely concerned. Currently, research on bacterial detoxification mainly focuses on groups such as Lactobacillus.spp, Bacillus.spp, Brevibacterium.spp and Acinetobacter.spp. The main manifestation of the detoxification effect is the adsorption and degradation of OTA.

[0004] In addition, degradation is the most important aspect of biological detoxification of ochratoxin. The specific degradation of toxins by microorganisms or enzymes secreted by microorganisms to obtain non-toxic or low-toxic degradation products. At present, certain progress has been made in the biological detoxification of ochratoxin using microorganisms, but there is still less research on ochratoxin degrading enzymes, especially the industrial application of ochratoxin degrading enzymes.

[0005] Currently, the effect of commercial ochratoxin degrading enzymes is poor and cannot meet industrial applications. Therefore, it is necessary to provide strains and degrading enzymes with high efficiency in degrading OTA and inhibiting OTA-producing fungi to solve the above problems. Summary of the Invention

[0006] One object of the present invention is to provide a strain of Brevibacillus brevis and its application in degrading ochratoxin A (OTA).

[0007] Another object of the present invention is to provide an OTA degrading enzyme and its application in degrading OTA.

[0008] To achieve the above object, the present invention adopts the following technical solutions:

[0009] The present invention first provides a strain of Brevibacillus, with the preservation number of CGMCC No. 25673, classified and named as Brevibacillus sp., and deposited in the General Microbiology Center of the China Center for Type Culture Collection (Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing) on September 9, 2022.

[0010] Furthermore, the present invention also provides a microbial preparation, the active ingredient of which comprises the above-mentioned Brevibacillus or its fermentation broth or its cell resuspension or its culture or its metabolite.

[0011] Furthermore, the fermentation broth of the Brevibacillus is obtained by inoculating the above-mentioned Brevibacillus into an LB liquid medium for culture.

[0012] Furthermore, the cell resuspension of the Brevibacillus is obtained by inoculating the above-mentioned Brevibacillus into an LB liquid medium for culture, centrifuging, and then resuspending the cells.

[0013] The Brevibacillus strain ASAG55 of the present invention is screened, isolated and purified from a wheat sample, and is identified as Brevibacillus sp. through morphological characteristics and 16S rDNA gene sequence analysis. The nucleotide sequence of the 16S rDNA of this strain is as shown in SEQ ID NO.1. The colony morphology is round, convex on the surface, and opaque; the cells are rod-shaped; and the Gram stain is positive.

[0014] The present invention further provides an OTA degrading enzyme, and the OTA degrading enzyme is A1-01 or A1-05. The amino acid sequence of A1-01 is shown as A1) or A2) or A3) or A4):

[0015] A1) A protein with the amino acid sequence shown in SEQ ID NO.4;

[0016] A2) A fusion protein obtained by connecting a protein tag to the N-terminus or C-terminus of the amino acid sequence shown in SEQ ID NO.4;

[0017] A3) A product obtained by performing conventional modifications on the side chain groups and / or N-terminus and / or C-terminus of the amino acid sequence shown in SEQ ID NO.4;

[0018] A4) A protein obtained by substituting and / or deleting and / or adding one or several amino acid residues to the amino acid sequence shown in SEQ ID NO.4, which has more than 90% identity and the same function as the protein shown in A1);

[0019] The amino acid sequence of A1-05 is as shown in B1) or B2) or B3) or B4):

[0020] B1) A protein with the amino acid sequence shown in SEQ ID NO.5;

[0021] B2) A fusion protein obtained by connecting a protein tag to the N-terminus or C-terminus of the amino acid sequence shown in SEQ ID NO.5;

[0022] B3) A product obtained by performing conventional modifications on the side chain groups and / or N-terminus and / or C-terminus of the amino acid sequence shown in SEQ ID NO.5;

[0023] B4) A protein obtained by substituting and / or deleting and / or adding one or several amino acid residues to the amino acid sequence shown in SEQ ID NO.5, having more than 90% identity with the protein shown in B1) and having the same function.

[0024] Furthermore, the protein tag can be a Flag tag, His tag, MBP tag, HA tag, myc tag, GST tag, and / or SUMO tag, etc.; the conventional modifications are amination, amidation, hydroxylation, carboxylation, carbonylation, alkylation, acetylation, phosphorylation, esterification, glycosylation, cyclization, ubiquitination, or biotinylation, etc.; the identity of more than 90% can be at least 91%, 92%, 95%, 96%, 98%, 99%, or 100% identity.

[0025] Furthermore, the gene encoding the above OTA degrading enzyme is also within the protection scope of the present invention. The nucleotide sequence of the gene of A1-01 is as shown in SEQ ID NO.2, and the nucleotide sequence of the gene of A1-05 is as shown in SEQ ID NO.3.

[0026] Furthermore, the primer pairs for amplifying the gene of the above OTA degrading enzyme are also within the protection scope of the present invention. The nucleotide sequences of the primer pairs for the gene of A1-01 are as shown in SEQ ID NO.6 and SEQ ID NO.7 or SEQ ID NO.10 and SEQ ID NO.11, and the nucleotide sequences of the primer pairs for the gene of A1-05 are as shown in SEQ ID NO.8 and SEQ ID NO.9 or SEQ ID NO.12 and SEQ ID NO.13.

[0027] Furthermore, an expression cassette, recombinant vector, or recombinant microorganism containing the gene of the above OTA degrading enzyme is also within the protection scope of the present invention.

[0028] Furthermore, the expression cassette refers to DNA containing the gene capable of expressing the OTA degrading enzyme in a host cell. This DNA may not only include the promoter that initiates the transcription of the OTA degrading enzyme gene, but also include the terminator that terminates the transcription of the OTA degrading enzyme gene. Further, the expression cassette may also include enhancer sequences.

[0029] Furthermore, the recombinant vector is a vector containing the gene expression cassette of the OTA degrading enzyme, and the vector can be pET-28a(+) or pPIC9K.

[0030] In a specific embodiment of the present invention, the recombinant vector can be the recombinant plasmid pET-28a(+) / A1-01 or pET-28a(+) / A1-05. Among them, the recombinant plasmid pET-28a(+) / A1-01 is a recombinant plasmid obtained by replacing the DNA fragment between the BamH I and Xho I recognition sequences of the pET-28a(+) plasmid with the A1-01 gene shown in SEQ ID NO.2 while keeping other sequences unchanged. The recombinant plasmid pET-28a(+) / A1-05 is a recombinant plasmid obtained by replacing the DNA fragment between the BamH I and Xho I recognition sequences of the pET-28a(+) plasmid with the A1-05 gene shown in SEQ ID NO.3 while keeping other sequences unchanged.

[0031] The recombinant vector can also be the recombinant yeast expression plasmid pPIC9K-A1-01 or pPIC9K-A1-05. Among them, the recombinant yeast expression plasmid pPIC9K-A1-01 is a recombinant plasmid obtained by replacing the DNA fragment between the SnaB I and Not I recognition sequences of the pPIC9K plasmid with the A1-01 gene shown in SEQ ID NO.2 in the sequence listing while keeping other sequences unchanged. The recombinant yeast expression plasmid pPIC9K-A1-05 is a recombinant plasmid obtained by replacing the DNA fragment between the SnaB I and Not I recognition sequences of the pPIC9K plasmid with the A1-05 gene shown in SEQ ID NO.3 in the sequence listing while keeping other sequences unchanged.

[0032] Furthermore, the host of the recombinant microorganism can be fungi and bacteria. Among them, the fungi can be yeast, specifically the Pichia pastoris strain GS115, and the bacteria can be Escherichia coli, specifically Escherichia coli Rosetta(DE3).

[0033] In a specific embodiment of the present invention, the recombinant microorganism is recombinant strain Rosetta[pET-28a(+) / A1-01] or Rosetta[pET-28a(+) / A1-05], wherein the recombinant strain Rosetta[pET-28a(+) / A1-01] is a recombinant strain obtained by transferring the recombinant plasmid pET-28a(+) / A1-01 into Escherichia coli Rosetta(DE3) competent cells, and the recombinant strain Rosetta[pET-28a(+) / A1-05] is a recombinant strain obtained by transferring the recombinant plasmid pET-28a(+) / A1-05 into Escherichia coli Rosetta(DE3) competent cells;

[0034] The recombinant microorganism can also be recombinant Pichia pastoris strain GS115[pPIC9K-A1-01] or GS115[pPIC9K-A1-05], wherein the recombinant Pichia pastoris strain GS115[pPIC9K-A1-01] is a recombinant strain obtained by transferring the recombinant yeast expression plasmid pPIC9K-A1-01 into Pichia pastoris GS115, and the recombinant Pichia pastoris strain GS115[pPIC9K-A1-05] is a recombinant strain obtained by transferring the recombinant yeast expression plasmid pPIC9K-A1-05 into Pichia pastoris GS115.

[0035] The present invention further provides the following applications of the above-mentioned Brevibacillus brevis, the above-mentioned microbial preparation, the above-mentioned OTA degrading enzyme, the gene containing the above-mentioned OTA degrading enzyme, the expression cassette containing the gene of the above-mentioned OTA degrading enzyme, the recombinant vector or the recombinant microorganism in any of the following:

[0036] C1) Application in degrading OTA in a sample;

[0037] C2) Application in preparing a product for degrading OTA in a sample.

[0038] The present invention also provides a method for degrading OTA, comprising the following steps:

[0039] Adding the above-mentioned Brevibacillus brevis or the above-mentioned microbial preparation to a sample containing OTA, culturing to achieve the degradation of OTA in the sample;

[0040] Adding the above-mentioned OTA degrading enzyme to a sample containing OTA, reacting to achieve the degradation of OTA in the sample.

[0041] Further, the culturing conditions are incubation at 18-40 °C for 10-60 hours;

[0042] Further, the reaction conditions are reaction at 20-45 °C for 0.5-36 h.

[0043] In the present invention, the sample may be grains and their processing by-products, feeds, foods, etc.

[0044] The Brevibacillus strain of the present invention has a good degradation effect on OTA. After incubation at 30 °C for 48 h, the degradation rate of the cell resuspension on OTA reaches more than 97%. The OTA-degrading enzyme of the present invention has a good degradation effect on OTA. After reacting at 37 °C for 1 h, the degradation rate on OTA reaches 100%.

[0045] The beneficial effects of the present invention are as follows:

[0046] The Brevibacillus strain ASAG55 of the present invention and two OTA-degrading enzymes mined from the Brevibacillus strain ASAG55 have the functions of efficiently degrading OTA and biological control of OTA, and have good application prospects in the fields of OTA degradation and prevention and control. Description of the Drawings

[0047] The following further details the specific embodiments of the present invention in conjunction with the drawings.

[0048] Figure 1 It is the liquid chromatography diagram before and after OTA degradation.

[0049] Figure 2 It is the morphological characteristic diagram of the strain ASAG55; among them, (a) is the morphological characteristic of the strain ASAG55 on the LB medium, and (b) is the morphological characteristic of the strain ASAG55 under the microscope.

[0050] Figure 3 It is the degradation effect diagram of the cell resuspension of the strain ASAG55 on OTA.

[0051] Figure 4 It is the electrophoresis map of double enzyme digestion verification of the recombinant plasmids pET-28a(+) / A1-01 and pET-28a(+) / A1-05 of Escherichia coli; among them, lane 1 is Marker; lane 2 is the empty plasmid pET-28a(+); lane 3 is the product of double enzyme digestion of pET-28a(+) / A1-01 with BamHI / XhoI; lane 4 is the PCR identification of the recombinant of the OTA-degrading enzyme A1-05; lane 5 is the product of double enzyme digestion of pET-28a(+) / A1-05 with BamHI / XhoI; lane 6 is the PCR identification of the recombinant of the OTA-degrading enzyme A1-05.

[0052] Figure 5To verify the expression of OTA-degrading enzymes A1-01 and A1-05 by SDS-PAGE; among them, in (a), lane 1 is the protein Marker, lane 2 is the crude enzyme solution of A1-01, and lane 3 is the pure enzyme solution of A1-01; in (b), lane 1 is the protein Marker, lane 2 is the crude enzyme solution of A1-05, and lane 3 is the pure enzyme solution of A1-05.

[0053] Figure 6 It is the degradation effect diagram of OTA when OTA-degrading enzymes A1-01 and A1-05 react with the enzyme activity reaction system containing OTA for 30 min and 80 min respectively.

[0054] Figure 7 It is the map of the recombinant yeast expression plasmid; among them, (a) is pPIC9k-A1-01, and (b) is pPIC9k-A1-05.

[0055] Figure 8 It is the degradation effect diagram of OTA by OTA-degrading enzymes A1-01 and A1-05. Detailed implementation manners

[0056] To illustrate the present invention more clearly, the present invention will be further described below in conjunction with preferred embodiments and the accompanying drawings. Similar components in the drawings are represented by the same reference numerals. Those skilled in the art should understand that the content specifically described below is illustrative rather than restrictive, and should not be used to limit the protection scope of the present invention.

[0057] The formulations of the culture media and buffers used in the following examples are as follows:

[0058] Basal salt medium (MSM medium): (NH4)2SO4 0.5 g, MgSO4 0.2 g, Na2HPO4·12H2O 6.15 g, KH2PO4 1.52 g, CaCl2 0.05 g, add water to make up to 1 L, adjust the pH to 7.0, sterilize at 121 °C for 20 min and then reserve for use.

[0059] Potato dextrose agar medium (PDA medium): Potato leaching powder 10 g, glucose 20 g, add water to make up to 1 L. When dispensing, add 1.5 - 2% agar to each bottle, sterilize at 115 °C for 20 min and then reserve for use.

[0060] LB liquid medium: Tryptone 10 g, yeast extract 5 g, sodium chloride 10 g, add water to make up to 1 L, sterilize at 121 °C for 20 min and then reserve for use.

[0061] LB solid medium: After dispensing the LB liquid medium, add 1.5 - 2% agar to each bottle, sterilize at 121 °C for 20 min and then reserve for use.

[0062] BMGY medium: Peptone 20 g / L, yeast extract 10 g / L, glycerol 1%, K2HPO4 3.01 g / L, KH2PO4 11.8 g / L, sterilized at 121 °C for 20 min, then add filter-sterilized YNB and biotin to make the final concentrations 13.4 g / L and 4×10 -4 g / L respectively, mix well before use.

[0063] BMMY medium: Peptone 20 g / L, yeast extract 10 g / L, K2HPO4 3.01 g / L, KH2PO4 11.8 g / L, sterilized at 121 °C for 20 min, then add filter-sterilized YNB and biotin to make the final concentrations 13.4 g / L and 4×10 -4 g / L respectively, mix well before use.

[0064] MD solid medium: Prepare 20 g / L glucose solution, dispense into small bottles, add 1.5 - 2% agar powder, sterilize at 115 °C for 20 min, then add filter-sterilized YNB and biotin to make the final concentrations 13.4 g / L and 4×10 -4 g / L respectively, mix evenly before use.

[0065] YPD solid medium: After dispensing YPD liquid medium, add 1.5 - 2% agar powder and sterilize at 115 °C for 20 min.

[0066] Buffer formulation:

[0067] Formulation of phosphate buffered saline (PBS) (1 L): NaCl 8 g, KCl 0.2 g, Na2HPO4·12H2O 3.63 g, KH2PO4 0.24 g, pH = 7.4.

[0068] Phosphate buffer (PB): Mix 0.05 mol / L Na2HPO4 with 0.05 mol / L NaH2PO4 and adjust to the target pH value.

[0069] Example 1 Isolation, screening and identification of OTA-degrading strain ASAG55

[0070] I. Isolation and screening of strains

[0071] After eluting the collected microorganisms by shaking with sterile normal saline, 50 μL of the washing solution was taken and added to the MSM medium containing 2.5 μg of OTA to make the final system 500 μL. It was cultured in a shaker at 30 °C and 200 r / min for 7 days. Then, 100 μL was taken from the culture system and added to a new MSM medium containing 2.5 μg of OTA to make the final system 500 μL to obtain an enriched sample. The enriched sample was cultured in a shaker at 30 °C and 200 r / min for 7 days, and the change in the OTA content in the sample was detected by high-performance liquid chromatography (HPLC). For the samples capable of degrading OTA, 100 μL of the culture solution was taken and successively diluted 10 -2 、10 -3 、10 -4 times with sterile normal saline. Then, 100 μL was taken and spread on a plate containing LB solid medium and cultured in an incubator at 30 °C. Single colonies on the spread plate were picked and inoculated into 500 μL of MSM medium containing 5 μg / mL of OTA, and co-incubated at 30 °C and 220 r / min for 7 days to obtain a reaction solution. The reaction solution was fully shaken and mixed with an equal volume of methanol, centrifuged at 10000 rpm for 10 min, and filtered through a 0.22 μm filter membrane. This treatment was the experimental group. The control group was to co-incubate 500 μL of MSM medium containing 5 μg / mL of OTA at 30 °C and 220 r / min for 7 days to obtain a reaction solution, fully shake and mix the reaction solution with an equal volume of methanol, centrifuge at 10000 rpm for 10 min, and filter through a 0.22 μm filter membrane. The residual amount (content) of OTA in each group was detected by HPLC to determine the degradation effect of the strain on OTA.

[0072] Among them, the conditions for detecting the residual amount (content) of OTA by HPLC: using a C18 chromatographic column (250 mm × 4.6 mm), the mobile phase was A:B = 1:1, A was glacial acetic acid - water (2 + 100), B was acetonitrile, the detection wavelength: excitation wavelength 333 nm, emission wavelength 460 nm, injection volume 10 μL, flow rate 1 mL / min, and column temperature 30 °C.

[0073] OTA degradation rate (%) = (OTA content in the control group - OTA content in the experimental group) / OTA content in the control group × 100%.

[0074] One strain with high OTA degradation effect was obtained through comparison and named ASAG55. The results were as Figure 1 shown. After 7 days of co-incubation, the degradation rate of OTA by strain ASAG55 was 99.20%, indicating that the strain isolated in the present invention has the ability to degrade OTA. After separating the strain, it was identified and analyzed.

[0075] II. Identification and analysis of strain ASAG55

[0076] The morphological and physiological / biochemical characteristics of strain ASAG55 were identified according to the methods described in "Bergey's Manual of Determinative Bacteriology" (8th Edition). The specific results are as follows:

[0077] 1. Morphological characteristics: As shown in (a) and (b) below, the colony morphology of strain ASAG55 on LB medium is round, convex on the surface, and opaque; Gram staining is positive, and the cells are rod-shaped under the microscope. Figure 2 As shown in (a) and (b) below, the colony morphology of strain ASAG55 on LB medium is round, convex on the surface, and opaque; Gram staining is positive, and the cells are rod-shaped under the microscope.

[0078] 2. Strain ASAG55 has the following biological properties: It can utilize mannitol, glucose, maltose, sucrose, and rhamnose, but cannot utilize sorbitol and arabinose.

[0079] 3. 16S rDNA gene identification:

[0080] The bacterial genomic DNA of strain ASAG55 was extracted and used as a template. The 16S rDNA gene of the bacteria was amplified by PCR using the universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-TACGGCTACCTTGTTACGACTT-3') for bacteria. Among them, the PCR amplification program was: pre-denaturation at 94°C for 5 min, denaturation at 94°C for 1 min, annealing at 54°C for 1 min, extension at 72°C for 2 min, and 30 cycles. The PCR amplification system was: 1 μL of 27F, 1 μL of 1492R, 1 μL of template, 25 μL of Taq enzyme, and 22 μL of ddH2O.

[0081] The PCR product was detected by 1% agarose gel, cut, recovered, purified, and then sequenced and analyzed. The nucleotide sequence of the 16S rDNA of strain ASAG55 is shown as SEQ ID NO.1, with a full length of approximately 1500 bp. After BLAST alignment and analysis in Genbank, this bacterium was identified as Brevibacillus sp.

[0082] The above identification results indicate that the strain ASAG55 belongs to the genus Brevibacillus, which belongs to the family Brevibacillaceae and the genus Brevibacillus. For further confirmation, the strain ASAG55 was sent to the China General Microbiological Culture Collection Center (abbreviated as CGMCC) in September 2021 for identification. ASAG55 was identified as the genus Brevibacillus sp. The strain ASAG55 was deposited in the China General Microbiological Culture Collection Center (abbreviated as CGMCC, address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, China, postal code: 100101) on September 9, 2022. Its deposit number is CGMCC No. 25673, and the taxonomic name is Brevibacillus sp.

[0083] Example 2 Degradation effect of strain ASAG55 on OTA

[0084] Pick a single colony of strain ASAG55 into 5 mL of LB liquid medium and culture it overnight on a shaker at 30 °C to obtain the fermentation broth of strain ASAG55. The fermentation broth was centrifuged at 8000 r / min for 20 min to remove the supernatant, washed twice with sterile saline, and then the cells were resuspended with saline to obtain the cell suspension of strain ASAG55.

[0085] The cell suspension of strain ASAG55 (final concentration of 1×10 7 CFU / mL) was inoculated into 5 mL of MSM medium containing OTA (final concentration of OTA was 5 μg / mL) at an inoculation amount of 5%. Incubate at 30 °C and 220 r / min for 0, 6, 12, 18, 24, 32, 40, 48, 56, 64, 72, 80, 88, 96 hours to obtain the reaction solution. The reaction solution was mixed thoroughly with an equal volume of methanol, centrifuged at 10000 rpm for 10 min, and filtered through a 0.22 μm filter membrane. This treatment was the experimental group. The control group was to incubate 5 mL of MSM medium containing OTA (final concentration of OTA was 5 μg / mL) at 30 °C and 220 r / min for 0, 6, 12, 18, 24, 32, 40, 48, 56, 64, 72, 80, 88, 96 hours to obtain the reaction solution. The reaction solution was mixed thoroughly with an equal volume of methanol, centrifuged at 10000 rpm for 10 min, and filtered through a 0.22 μm filter membrane. The residual amount (content) of OTA in each group was detected by HPLC method, and the method was the same as that in Example 1.

[0086] The results are as Figure 3 shown. After incubation for 24 h, the degradation rate of OTA by the cell suspension of strain ASAG55 was greater than 92%. After incubation for 48 h, the degradation rate of OTA by the cell suspension was greater than 98%, indicating that strain ASAG55 can efficiently degrade OTA.

[0087] Cloning, Construction, Expression and Functional Verification of Degrading Genes in Strain ASAG55 of Example 3

[0088] I. Cloning of OTA Degrading Enzyme Genes

[0089] Through gene discovery, two novel OTA degrading enzymes were screened and named A1-01 and A1-05 respectively. Their nucleotide sequences are shown in SEQ ID NO.2 and SEQ ID NO.3, and their amino acid sequences are shown in SEQ ID NO.4 and SEQ ID NO.5. The upstream primers and downstream primers of the two genes are respectively:

[0090] A1-01-F:

[0091] A1-01-R:

[0092] A1-05-F:

[0093] A1-05-R:

[0094] Restriction enzyme cleavage sites of BamH I and Xho I were added to the upstream and downstream primers respectively (the restriction enzymes are underlined and the protection bases are italic sequences).

[0095] The bacterial genomic DNA of strain ASAG55 was extracted and used as a template. Using A1-01-F and A1-01-R, A1-05-F and A1-05-R as primers respectively, PCR products containing OTA degrading enzyme genes A1-01 and A1-05 were amplified.

[0096] II. Construction and Expression

[0097] The PCR products containing OTA degrading enzyme genes A1-01 and A1-05 and the Escherichia coli expression vector pET-28a(+) were double digested with BamH I and Xho I respectively. The enzyme digestion system for the PCR products containing OTA degrading enzyme gene A1-01 or A1-05 was 150 μL, specifically as follows: 6.5 μL of the PCR product containing OTA degrading enzyme gene A1-01, 15 μL of 10× Buffer, 5 μL of BamH I, 5 μL of XhoI, and 118.5 μL of ddH2O; or, 8.5 μL of the PCR product containing OTA degrading enzyme gene A1-05, 15 μL of 10× Buffer, 5 μL of BamH I, 5 μL of Xho I, and 116.5 μL of ddH2O. The enzyme digestion system for the Escherichia coli expression vector pET-28a(+) was 160 μL, specifically as follows: 48 μL of pET-28a(+), 16 μL of 10×H buffer, 8 μL of BamH I, 8 μL of Xho I, and 80 μL of ddH2O. Both were double digested with Xho I and BamH I at 37°C for 3 h. After recovery by agarose gel electrophoresis, the double digested products of A1-01 gene, A1-05 gene, and the expression vector pET-28a(+) were obtained respectively. The products were ligated, and the ligation system was as follows: 1.0 μL of the double digested product of the expression vector pET-28a(+), 1.1 μL of the double digested product of A1-01 gene, 5.4 μL of ddH2O, and 7.5 μL of Solution I; or, 1.0 μL of the double digested product of the expression vector pET-28a(+), 1.2 μL of the double digested product of A1-05 gene, 5.3 μL of ddH2O, and 7.5 μL of Solution I. Ligation was carried out overnight at 16°C. The ligation products were transformed into Escherichia coli DH5α competent cells. After overnight culture, transformants were picked and plasmids were extracted for double digestion verification and sequencing verification. The results of double digestion verification using restriction endonucleases BamH I and Xho I were as Figure 4As shown, it can be seen from the figure that after double digestion of plasmids pET-28a(+) / A1-01 and pET-28a(+) / A1-05 respectively, the sizes of the target fragments are consistent with those of the PCR fragments of genes A1-01 and A1-05, and the sequencing results show that the sequences of the target fragments in pET-28a(+) / A1-01 and pET-28a(+) / A1-05 are correct, indicating successful construction of the recombinant vectors. The transformants with correct enzyme digestion verification and sequencing verification were used to extract plasmids respectively, and the recombinant plasmids pET-28a(+) / A1-01 and pET-28a(+) / A1-05 carrying the degradation enzyme genes A1-01 and A1-05 were obtained. The recombinant plasmids pET-28a(+) / A1-01 and pET-28a(+) / A1-05 were respectively transferred into Escherichia coli Rosetta(DE3) competent cells to construct two recombinant strains Rosetta[pET-28a(+) / A1-01] and Rosetta[pET-28a(+) / A1-05] of the degradation enzyme genes A1-01 and A1-05.

[0098] The two recombinant strains Rosetta[pET-28a(+) / A1-01] and Rosetta[pET-28a(+) / A1-05] were cultured overnight with shaking at 37 °C in 5 mL of LB liquid medium containing 50 μg / mL kanamycin. 1% of the above culture solution was transferred to 60 mL of LB liquid medium containing 50 μg / mL kanamycin and cultured at 37 °C and 200 r / min until the OD600 reached 0.6, then IPTG with a final concentration of 0.8 mM was added and induced at 18 °C for 18 hours. The cells were collected by centrifugation at 7000 r / min for 10 min at 4 °C, resuspended with 6 mL of PBS, sonicated on ice bath, and then centrifuged at 12000 r / min for 20 min at 4 °C to collect the supernatant, and crude enzyme solutions of two OTA degradation enzymes A1-01 and A1-05 were obtained respectively.

[0099] 20 mL of the crude enzyme solution was purified using a Ni column with PBS as the equilibration buffer. After collecting the sample penetration solution, elution was carried out successively with imidazole solutions of increasing concentrations, and the imidazole elution samples at each concentration were collected. SDS-PAGE electrophoresis was performed using the above collected samples and known-concentration bovine serum albumin samples. The SDS-PAGE electrophoresis results showed that the target protein was contained in the supernatant of cell disruption. The purified samples of crude enzyme solutions of A1-01 and A1-05 were eluted under the condition of 500 mM imidazole concentration, and pure enzyme solutions of A1-01 and A1-05 were obtained and their concentrations were determined. The theoretical molecular sizes of the two OTA degradation enzymes A1-01 and A1-05 are 43.3 kDa and 42.9 kDa respectively, and the SDS-PAGE analysis results are as Figure 5As shown in (a) and (b), in (a), by comparing with the Marker in Lane 1, it can be clearly seen that the target protein in Lane 3 is about 43 kDa, that is, similar in size to OTA degrading enzyme A1-01; in (b), by comparing with the Marker in Lane 1, it can be clearly seen that the target protein in Lane 3 is less than 50 kDa, significantly greater than 37 kDa, about 43 kDa, that is, similar in size to OTA degrading enzyme A1-05.

[0100] III. Function verification

[0101] PBS, the lysate of Rosetta(DE3) containing pET-28a(+) empty plasmid, and the crude enzyme solutions of A1-01 and A1-05 were respectively added to a 500 μL enzyme activity reaction system. The enzyme activity reaction system contained 10 μL of anhydrous ethanol containing 1 μg of OTA. The addition amount of the enzyme solution was 10 μg of A1-01 enzyme and 0.1 μg of A1-05 enzyme, and the remaining volume was added with PBS (pH 7.4) to make the total volume 500 μL (the final concentration of OTA was 2 μg / mL). After reacting at 37 °C for 60 min and 30 min respectively, 500 μL of methanol was added to terminate the reaction. The supernatant was taken by centrifugation, and the residual amount (content) of OTA was detected by HPLC method, and the method was the same as that in Example 1. The OTA content detected by adding PBS was used as the OTA content of the control group, and the OTA contents detected by adding the lysate of Rosetta(DE3) containing pET-28a(+) empty plasmid, and the crude enzyme solutions of A1-01 and A1-05 were used as the OTA contents of the experimental groups respectively to calculate the OTA degradation rate (%).

[0102] The results are as Figure 6 shown. The results show that after the enzyme activity reaction system reacted for 60 min and 30 min respectively, compared with the control group added with PBS buffer, the degradation rates of the two crude enzyme solutions (represented by "A1-01" and "A1-05" in the figure) to OTA could reach 100%. And the lysate of Rosetta(DE3) containing pET-28a(+) empty plasmid (represented by "CK-pET-28a(+)" in the figure) was confirmed to have no degradation activity to OTA.

[0103] Construction and expression of degrading enzyme in Pichia pastoris in Example 4

[0104] I. Cloning of OTA degrading enzyme gene

[0105] The bacterial genomic DNA of strain ASAG55 was extracted. Using it as a template, PCR amplifications were respectively carried out with A1-01-F and A1-01-R, A1-05-F and A1-05-R as primers to obtain PCR products containing OTA degrading enzyme genes A1-01 and A1-05.

[0106] Among them,

[0107] A1-01-F:

[0108] A1-01-R:

[0109] A1-05-F:

[0110] A1-05-R:

[0111] Restriction enzyme cleavage sites of SnaB I and Not I are added to the upstream and downstream primers respectively (the restriction enzymes are underlined, and the protection bases are italic sequences).

[0112] II. Construction and expression

[0113] The PCR products containing the OTA degrading enzyme genes A1-01 or A1-05 are double digested with restriction enzymes SnaB I and Not I respectively. The 100 μL digestion system is as follows: 40 μL of the PCR product of the OTA degrading enzyme gene A1-01 or A1-05, 10 μL of 10×H buffer, 5 μL of SnaB I, 5 μL of Not I, and 40 μL of ddH2O. After digestion at 37°C for 4 h, agarose gel electrophoresis is used for recovery, and the double digested products of genes A1-01 and A1-05 are obtained respectively.

[0114] The expression vector pPIC9K is double digested with restriction enzymes SnaB I and Not I. The 100 μL digestion system is as follows: 20 μL of the vector pPIC9K, 10 μL of 10×H buffer, 5 μL of SnaB I, 5 μL of Not I, and 65 μL of ddH2O. After digestion at 37°C for 4 h, agarose gel electrophoresis is used for recovery, and the double digested product of the expression vector pPIC9K is obtained.

[0115] The double-digested products of genes A1-01 and A1-05 were respectively ligated with the double-digested product of the expression vector pPIC9K to construct recombinant yeast expression plasmids pPIC9K-A1-01 and pPIC9K-A1-05. The ligation system was as follows: 5 μL of the double-digested product of the expression vector pPIC9K, 3 μL of the double-digested product of gene A1-01 or A1-05, and 8 μL of Solution I. Ligation was carried out overnight at 16 °C. The ligation products were transformed into Escherichia coli DH5α, and the transformants were picked for sequencing verification. The transformants with correct sequencing verification were transferred to LB (containing 50 μg / mL kanamycin) liquid medium and cultured overnight at 37 °C. Plasmids were extracted to obtain recombinant yeast expression plasmids pPIC9K-A1-01 and pPIC9K-A1-05 respectively (the plasmid maps are as Figure 7 shown).

[0116] The recombinant yeast expression plasmids pPIC9K-A1-01 and pPIC9K-A1-05 were linearized with BspE I and Sal I respectively. After purification of the linearized products, they were transformed into Pichia pastoris GS115 by electroporation and spread on MD plates. The colonies grown on the MD plates were Pichia pastoris engineering strains, namely recombinant Pichia pastoris strains GS115[pPIC9K-A1-01] and GS115[pPIC9K-A1-05]. Then they were spread on YPD plates containing different concentrations of geneticin G418 to screen for multi-copy transformants.

[0117] Example 5 Experiment on the Degradation Effect of Recombinant Enzyme on OTA

[0118] Single multi-copy transformants were picked and inoculated into BMGY medium respectively. After shaking culture at 30 °C and 220 r / min for 24 hours, they were transferred to BMMY medium and cultured with shaking at 30 °C and 220 r / min. 0.5% methanol was supplemented every 24 h. Continuous induction expression was carried out for 5 d. At the same time, samples were collected, and the cells were removed by centrifugation to obtain the supernatant, namely the crude enzyme solutions of recombinant enzymes A1-01 and A1-05, for in vitro OTA degradation experiments. The degradation system contained 1 μg OTA, 100 μL of the crude enzyme solution of recombinant enzyme A1-01 or 10 μL of the crude enzyme solution of recombinant enzyme A1-05, and PBS (pH 7.4) was added to make up to 500 μL (the final concentration of OTA was 2 μg / mL). Samples were taken every 20 min within 60 min of reaction at 37 °C, and the reaction was terminated with methanol. The supernatant was taken after centrifugation. This treatment was set as the experimental group. The treatment of replacing the crude enzyme solution of recombinant enzyme A1-01 or the crude enzyme solution of recombinant enzyme A1-05 with the fermentation supernatant of Pichia pastoris GS115 containing the pPIC9K empty plasmid was set as the control group. The change in OTA content was detected by HPLC method, and the method was the same as that in Example 1. The results are as Figure 8As shown, the results indicate that, compared with the control group (denoted as "CK-pPIC9K" in the figure) adding the fermentation supernatant of Pichia pastoris GS115 containing the empty plasmid pPIC9K, the degradation rates of OTA by the two crude enzyme solutions (denoted as "A1-01" and "A1-05" in the figure) can both reach 100%.

[0119] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, rather than limitations on the implementation manners of the present invention. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description. It is impossible to list all the implementation manners here. Any obvious changes or modifications derived from the technical solutions of the present invention still fall within the protection scope of the present invention.

Claims

1. A Brevibacillus strain ( Brevibacillus sp. ), characterized in that The preservation number of the Brevibacillus is CGMCC No. 25673.

2. A microbial preparation, characterized in that, The active ingredient of the microbial preparation comprises the Brevibacillus described in claim 1, or its fermentation broth, or its cell resuspension, or its culture.

3. Use of the Brevibacillus described in claim 1 or the microbial preparation described in claim 2 in any of the following: C1) Use in degrading ochratoxin A in a sample; C2) Use in preparing a product for degrading ochratoxin A in a sample.

4. Use of an ochratoxin A degrading enzyme or a gene encoding the ochratoxin A degrading enzyme in any of the following: C1) Use in degrading ochratoxin A in a sample; C2) Use in preparing a product for degrading ochratoxin A in a sample; Among them, The ochratoxin A degrading enzyme is A1-01 or A1-05, and the amino acid sequence of A1-01 is shown as A1), A2), or A3): A1) A protein with the amino acid sequence shown in SEQ ID NO. 4; A2) A fusion protein obtained by linking a protein tag to the N-terminus or C-terminus of the amino acid sequence shown in SEQ ID NO. 4; A3) A product obtained by performing conventional modifications on the side chain groups and / or N-terminus and / or C-terminus of the amino acid sequence shown in SEQ ID NO. 4; the conventional modifications are amination, amidation, hydroxylation, carboxylation, carbonylation, alkylation, acetylation, phosphorylation, esterification, glycosylation, cyclization, ubiquitination, or biotinylation; The amino acid sequence of A1-05 is shown as B1), B2), or B3): B1) A protein with the amino acid sequence shown in SEQ ID NO. 5; B2) A fusion protein obtained by linking a protein tag to the N-terminus or C-terminus of the amino acid sequence shown in SEQ ID NO. 5; B3) A product obtained by performing conventional modifications on the side chain groups and / or N-terminus and / or C-terminus of the amino acid sequence shown in SEQ ID NO. 5; the conventional modifications are amination, amidation, hydroxylation, carboxylation, carbonylation, alkylation, acetylation, phosphorylation, esterification, glycosylation, cyclization, ubiquitination, or biotinylation; The nucleotide sequence of the gene of A1-01 is as shown in SEQ ID NO. 2, and the nucleotide sequence of the gene of A1-05 is as shown in SEQ ID NO.

3.

5. A method for degrading ochratoxin A, characterized in that, Comprising the following steps: Adding the Brevibacillus described in claim 1 or the microbial preparation described in claim 2 to a sample containing ochratoxin A, culturing, to achieve the degradation of ochratoxin A in the sample; Adding the ochratoxin A degrading enzyme to a sample containing ochratoxin A, reacting, to achieve the degradation of ochratoxin A in the sample; Wherein, the ochratoxin A degrading enzyme is A1-01 or A1-05, and the amino acid sequence of A1-01 is shown as A1), A2), or A3): A1) A protein with the amino acid sequence shown in SEQ ID NO. 4; A2) A fusion protein obtained by linking a protein tag to the N-terminus or C-terminus of the amino acid sequence shown in SEQ ID NO. 4; Products obtained by performing conventional modifications on the side chain groups and / or N-terminus and / or C-terminus of the amino acid sequence shown in SEQ ID NO. 4; the conventional modifications are amination, amidation, hydroxylation, carboxylation, carbonylation, alkylation, acetylation, phosphorylation, esterification, glycosylation, cyclization, ubiquitination or biotinylation; The amino acid sequence of A1-05 is as shown in B1) or B2) or B3): B1) A protein with the amino acid sequence shown in SEQ ID NO. 5; B2) A fusion protein obtained by linking a protein tag to the N-terminus or C-terminus of the amino acid sequence shown in SEQ ID NO. 5; B3) Products obtained by performing conventional modifications on the side chain groups and / or N-terminus and / or C-terminus of the amino acid sequence shown in SEQ ID NO. 5; the conventional modifications are amination, amidation, hydroxylation, carboxylation, carbonylation, alkylation, acetylation, phosphorylation, esterification, glycosylation, cyclization, ubiquitination or biotinylation.

6. The method according to claim 5, wherein The culture conditions are incubation at 18-40 °C for 10-60 hours; the reaction conditions are reaction at 20-45 °C for 0.5-36 h.

7. The application according to claim 3, the application according to claim 4, or the method according to claim 5, characterized in that The sample is grains and their processing by-products.

8. The application according to claim 3, the application according to claim 4 or the method according to claim 5, characterized in that, The sample is feed or food.

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