Pichia guilliermongi for removing aflatoxin, aflatoxin-degrading enzyme and application thereof

Abstract

The present invention discloses aflatoxin-removing Pichia guilliermong, aflatoxin-degrading enzyme and application thereof. The present invention first discloses Pichia guilliermong and application thereof in aflatoxin removal. The present invention further discloses aflatoxin-degrading enzyme and application thereof in aflatoxin removal. The Pichia guilliermong ASAG57 and aflatoxin-degrading enzyme mined from the Pichia guilliermong ASAG57 of the present invention have good aflatoxin removal effect, have a biocontrol function of aflatoxin, and have good application prospects in the field of aflatoxin removal and prevention and control.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, and more specifically to aflatoxin-removing Pichia guilliermong, aflatoxin-degrading enzymes and applications thereof. Background Art

[0002] Aflatoxin (AFT) is a highly toxic secondary metabolite produced by Aspergillus flavus and Aspergillus parasiticus. It is also the most carcinogenic toxin among known chemicals. Aflatoxin is widely present in food and feed. It is seriously contaminated in peanuts, corn and their processing by-products. Its pollution seriously threatens the health of humans and livestock and poultry, and also brings huge economic losses to grain and oil processing and feed breeding companies.

[0003] Aflatoxin has a stable structure and is not easily degraded under light, heat and acidic conditions. It is also difficult to be destroyed during conventional processing. Aflatoxin can be removed by adsorption, biodegradation, chemical degradation, photodegradation and other methods, but the most widely used and effective methods are adsorption and biodegradation. Some minerals and their modified products have a good adsorption effect on aflatoxin, but their adsorption is not specific. The adsorbent has a physical air barrier effect, and the adsorption effect has a certain limit. In addition, mineral adsorbents will increase the ash content of feed raw materials and feed, affecting palatability. Biological detoxification methods include microbial fermentation degradation, enzymatic degradation and biological adsorption. The biodegradation method has the advantages of specificity, high efficiency, no effect on the properties of the raw materials and environmental friendliness. The biological adsorption method can improve the nutritional level of the rate while removing aflatoxin. The biological detoxification method has a greater advantage and is more suitable for industrial applications, but the existing aflatoxin detoxification strains cannot meet the needs of industrial applications.

[0004] Therefore, it is necessary to provide a biological detoxification agent that can effectively remove aflatoxin to solve the above problems. Summary of the invention

[0005] One object of the present invention is to provide a strain of Pichia guilliermong and its application in removing aflatoxin.

[0006] Another object of the present invention is to provide an aflatoxin degrading enzyme and its application in removing aflatoxin.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions:

[0008] The present invention first provides a strain of Pichia guilliermondii, which has a preservation number of CGMCC No. 25722, is classified and named Meyerozyma guilliermondii, and was preserved in the General Biological Center of China National Microbiological Culture Collection Administration on September 15, 2022 (address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing).

[0009] Furthermore, the present invention also provides a microbial preparation, the active ingredients of which include the above-mentioned Pichia guilliermongo or its fermentation broth or its fermentation broth whole cell fragmentation liquid or its inactivated fermentation broth whole cell fragmentation liquid or its proteinase K treated fermentation broth whole cell fragmentation liquid or its culture or its metabolites.

[0010] In a specific embodiment of the present invention, the Pichia quaternaria fermentation broth is obtained by inoculating the above-mentioned Pichia quaternaria into YPD liquid culture medium for cultivation.

[0011] In a specific embodiment of the present invention, the fermentation broth whole cell disruption liquid is obtained by ultrasonically disrupting the fermentation broth; the fermentation broth whole cell disruption liquid is obtained by inactivating it by heating at 121°C for 20 minutes; the fermentation broth whole cell disruption liquid is obtained by treating a proteinase K reagent with a final concentration of 1 mg / mL at 55°C for 1 hour.

[0012] The Pichia guilliermondii strain ASAG57 of the present invention is screened, separated and purified from contaminated grain samples, and is identified as Pichia guilliermondii through morphological characteristics and 18S rDNA gene sequence analysis. The nucleotide sequence of 18S rDNA of the strain is shown in SEQ ID NO.1.

[0013] The present invention further provides an aflatoxin degrading enzyme, wherein the amino acid sequence of the aflatoxin degrading enzyme is shown in A1) or A2) or A3) or A4):

[0014] A1) the amino acid sequence of the protein shown in SEQ ID No. 3;

[0015] 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. 3;

[0016] A3) a product obtained by performing conventional modifications on the side chain groups and / or the N-terminus and / or the C-terminus of the amino acid sequence shown in SEQ ID No. 3;

[0017] A4) A protein having more than 90% identity and the same function as the protein shown in A1) obtained by replacing and / or deleting and / or adding one or more amino acid residues in the amino acid sequence shown in SEQ ID No. 3.

[0018] Furthermore, the protein tag may be a Flag tag, a His tag, an MBP tag, an HA tag, a myc tag, a GST tag and / or a SUMO tag, etc.; the conventional modification may be amination, amidation, hydroxylation, carboxylation, carbonylation, alkylation, acetylation, phosphorylation, esterification, glycosylation, cyclization, ubiquitination or biotinylation, etc.; the identity of more than 90% may be at least 91%, 92%, 95%, 96%, 98%, 99% or 100% identity.

[0019] Furthermore, the gene encoding the above-mentioned aflatoxin degrading enzyme is also within the protection scope of the present invention, and the nucleotide sequence of the gene is shown in SEQ ID No.2.

[0020] Furthermore, a primer pair for amplifying the gene of the above-mentioned aflatoxin degrading enzyme is also within the protection scope of the present invention, and the nucleotide sequence of the primer pair is shown as SEQ ID NO.4 and SEQ ID NO.5 or SEQ ID NO.6 and SEQ ID NO.7.

[0021] Furthermore, expression cassettes, recombinant vectors or recombinant microorganisms containing the above-mentioned aflatoxin degrading enzyme genes are also within the protection scope of the present invention.

[0022] Furthermore, the expression cassette refers to a DNA capable of expressing the gene of aflatoxin degrading enzyme in a host cell, and the DNA may include not only a promoter for initiating transcription of the gene of aflatoxin degrading enzyme, but also a terminator for terminating transcription of the gene of aflatoxin degrading enzyme. Furthermore, the expression cassette may also include an enhancer sequence.

[0023] Furthermore, the recombinant vector is a vector containing a gene expression cassette of the aflatoxin degrading enzyme, and the vector may be pET-28a(+) or pPIC9K.

[0024] In a specific embodiment of the present invention, the recombinant vector can be a recombinant plasmid pET-28a(+)-MG2-4, wherein the recombinant plasmid pET-28a(+)-MG2-4 is a recombinant plasmid obtained by replacing the DNA fragment between the Sac I and Xho I recognition sequences of the pET-28a(+) plasmid with the MG2-4 gene shown in SEQ ID No. 2 in the sequence table and keeping other sequences unchanged;

[0025] The recombinant vector can also be a recombinant yeast expression plasmid pPIC9K+MG2-4, which 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 MG2-4 gene shown in SEQID No.2 in the sequence table and keeping other sequences unchanged.

[0026] Furthermore, the host of the recombinant microorganism may be a fungus or a bacterium, wherein the fungus may be a yeast, specifically a Pichia pastoris strain GS115, and the bacterium may be an Escherichia coli, specifically an Escherichia coli Rosetta (DE3).

[0027] In a specific embodiment of the present invention, the recombinant microorganism is a recombinant strain Rosetta [pET-28a (+) -MG2-4], and the recombinant strain Rosetta [pET-28a (+) -MG2-4] is a recombinant strain obtained by transferring the recombinant plasmid pET-28a (+) -MG2-4 into Escherichia coli Rosetta (DE3) competent cells;

[0028] The recombinant microorganism may also be a recombinant Pichia pastoris strain GS115[pPIC9K+MG2-4], wherein the recombinant Pichia pastoris strain GS115[pPIC9K+MG2-4] is a recombinant strain obtained by transferring the recombinant yeast expression plasmid pPIC9K+MG2-4 into Pichia pastoris GS115.

[0029] The present invention further provides the use of the above-mentioned Pichia guilliermongi or the above-mentioned microbial preparation or the above-mentioned aflatoxin degrading enzyme or the above-mentioned aflatoxin degrading enzyme gene or the above-mentioned aflatoxin degrading enzyme gene expression cassette, recombinant vector or recombinant microorganism in any of the following:

[0030] B1) Application in removing aflatoxin from samples;

[0031] B2) Application in the preparation of aflatoxin removal products in samples.

[0032] Furthermore, the sample may be grain and its processing by-products, feed, food, etc.

[0033] The present invention also provides a method for removing aflatoxin, comprising the following steps:

[0034] Adding the above-mentioned Pichia quaternaria or the above-mentioned microbial preparation to a sample containing aflatoxin, culturing, and achieving the removal of aflatoxin in the sample;

[0035] The aflatoxin degrading enzyme is added to a sample containing aflatoxin to react and remove the aflatoxin from the sample.

[0036] Furthermore, the culture conditions are 25-35°C and 50-300r / min for 0.5-12 hours.

[0037] Furthermore, the reaction conditions are 25-35° C. and 50-300 r / min for 0.5-12 hours.

[0038] The Pichia quaternaria of the present invention has good adsorption and degradation effects on aflatoxin, and the removal rate of aflatoxin reaches 95.55% after reacting for 1 hour at 30°C. The aflatoxin degrading enzyme of the present invention has good degradation effect on aflatoxin, and the degradation rate of aflatoxin reaches more than 89.6% after reacting for 1 hour at 30°C.

[0039] In the present invention, the aflatoxin is specifically aflatoxin B1 (AFB1).

[0040] In the present invention, the removal refers to degradation and / or adsorption.

[0041] The beneficial effects of the present invention are as follows:

[0042] The Pichia guilliermong ASAG57 of the present invention and the aflatoxin degrading enzyme mined from the Pichia guilliermong ASAG57 have good aflatoxin adsorption and degradation effects, have a biocontrol function of aflatoxin, and have good application prospects in the field of aflatoxin removal and prevention and control. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] The specific implementation modes of the present invention are further described in detail below in conjunction with the accompanying drawings.

[0044] Figure 1 This is the liquid chromatogram of a single colony of strain ASAG57 before and after aflatoxin removal after 7 days.

[0045] Figure 2 The colony morphology of strain ASAG57 on PDA medium.

[0046] Figure 3 The morphology of strain ASAG57 under an optical microscope.

[0047] Figure 4 The morphology of strain ASAG57 under scanning electron microscope.

[0048] Figure 5 This is the phylogenetic tree of strain ASAG57.

[0049] Figure 6This is a diagram showing the removal effect of each component of the fermentation broth of strain ASAG57 on aflatoxin.

[0050] Figure 7 It is the result of double enzyme digestion of the PCR product containing AFB1 degradation enzyme gene MG2-4 and Escherichia coli expression vector pET-28a(+); wherein, lane M is DNA Marker, lane 1 is the double enzyme digestion product of pET-28a(+), and lane 2 is the double enzyme digestion product of the PCR product containing AFB1 degradation enzyme gene MG2-4.

[0051] Figure 8 It is the colony PCR verification result of the positive transformant; Among them, lane M is the DNA Marker, and lane 1 is the target fragment of colony PCR.

[0052] Fig. 9 This is the map of the Escherichia coli recombinant plasmid pET-28a(+)-MG2-4.

[0053] Fig.10 It is an SDS-PAGE diagram of protein purification of the target gene expressed in Escherichia coli, wherein lane 1 is a crude enzyme solution, lane 2 is an enzyme solution eluted under 300mM imidazole concentration conditions, lane 3 is an enzyme solution eluted under 400mM imidazole concentration conditions, lane 4 is an enzyme solution eluted under 500mM imidazole concentration conditions, lane 5 is an enzyme solution eluted under the second 500mM imidazole concentration conditions, lane 6 is BSA 0.5mg / mL, and lane M is a marker.

[0054] Fig.11 This is the map of the recombinant yeast expression plasmid pPIC9K+MG2-4.

[0055] Fig.12 The figure is the liquid chromatogram of AFB1 degradation in vitro. DETAILED DESCRIPTION

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

[0057] The formulas of the culture medium and buffer used in the following examples are as follows:

[0058] Basal salt medium (MSM medium): (NH4)2SO4 0.5g, MgSO4 0.2g, Na2HPO412H2O 6.15g, KH2PO4 1.52g, CaCl2 0.05g, add water to make up to 1L, adjust the pH to 7.0, sterilize at 121℃ for 20min and set aside.

[0059] Potato dextrose agar medium (PDA medium): 10g potato extract powder, 20g glucose, add water to make up to 1L, add 1.5-2% agar to each bottle when packaging, sterilize at 115℃ for 20min and set aside.

[0060] Yeast extract peptone glucose medium (YPD liquid medium): 10g yeast extract, 20g peptone, 20g glucose, add water to make up to 1L, sterilize at 115℃ for 20min and set aside.

[0061] YPD solid culture medium: Add 1.5-2% agar powder to the YPD liquid culture medium and sterilize at 115℃ for 20 minutes.

[0062] LB medium: 10 g of tryptone, 5 g of yeast extract, 10 g of sodium chloride, add water to make up to 1 L, add 1.5-2% agar to each bottle when packaging, sterilize at 121°C for 20 min and set aside.

[0063] 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 degrees Celsius 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, mix well before use.

[0064] BMMY medium: peptone 20 g / L, yeast extract 10 g / L, K2HPO4 3.01 g / L, KH2PO4 11.8 g / L, sterilized at 121 degrees Celsius for 20 min, then filter-sterilized YNB and biotin were added to make the final concentrations 13.4 g / L and 4 × 10 -4 g / L, mix well before use.

[0065] MD solid medium: prepare 20g / L glucose solution, dispense into vials, add 1.5-2% agar powder, sterilize at 115℃ for 20min, add filter-sterilized YNB and biotin to make the final concentrations 13.4g / L and 4×10 -4 g / L, mix well before use.

[0066] Buffer formulation:

[0067] Phosphate buffered saline (PBS) formula (1L): NaCl 8g, KCl 0.2g, NA2HPO4 12H2O 3.63g, KH2PO4 0.24g, pH=7.4.

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

[0069] Example 1 Isolation, screening and identification of aflatoxin-removing strain ASAG57

[0070] 1. Isolation and screening of aflatoxin-removing strain ASAG57

[0071] Select contaminated grain samples, take 5g of the sample and add it to 30mL of sterile physiological saline, shake and mix, so that the microorganisms in the sample are fully suspended in the water. After standing for 30 minutes, take 50μL of the supernatant to MSM medium containing 1μg of aflatoxin B1 (AFB1), so that the final system is 500μL, 30℃, 220r / min constant temperature shaking culture for 7 days, and obtain the enrichment reaction liquid. Take 100μL of the enrichment reaction liquid and add 100μL of methanol to mix evenly, and detect the residual amount of AFB1 by high performance liquid chromatography (HPLC) to screen out samples with removal effect on AFB1. The samples with removal effect on AFB1 are diluted 10 -2 , 10 -3 , 10 -4 Then 100 μL was evenly spread on the MSM solid medium, and a single colony was picked and inoculated into 500 μL MSM medium containing 2 μg / mL AFB1. The reaction solution was obtained by co-incubation at 30°C and 220 r / min for 7 days. 100 μL of the reaction solution was added to 100 μL methanol and fully oscillated and mixed, centrifuged at 10000 r / min for 10 minutes, and filtered with a 0.22 μm filter membrane. This treatment was the experimental group. The control group was 500 μL MSM medium containing 2 μg / mL AFB1, co-incubated at 30°C and 220 r / min for 7 days to obtain the reaction solution. 100 μL of the reaction solution was added to 100 μL methanol and fully oscillated and mixed, centrifuged at 10000 r / min for 10 minutes, and filtered with a 0.22 μm filter membrane. The HPLC method was used to detect the residual amount (content) of AFB1 in each group and determine the removal effect of the strain on AFB1.

[0072] The conditions for HPLC detection of AFB1 residue (content) were as follows: the instrument used was Alliance e2695 (Waters, USA), a fluorescence detector was used, and a photochemical post-column derivatization was equipped. The excitation wavelength was 365 nm, and the emission wavelength was 450 nm. A C18 chromatographic column (250 mm × 4.6 mm) was used, the mobile phase was methanol: water (1:1, v / v), the flow rate was 0.5 mL / min, and the sample volume was 10 μL.

[0073] AFB1 removal rate (%) = (AFB1 content in control group - AFB1 content in experimental group) / AFB1 content in control group × 100;

[0074] By comparing the removal rates of AFB1 by colonies, a strain with high efficiency in removing AFB1 was screened and named ASAG57.

[0075] The results are as follows Figure 1 The results showed that after 7 days of co-incubation, the removal rate of AFB1 by strain ASAG57 was 85.90%, indicating that the strain ASAG57 isolated by the present invention has the ability to remove AFB1. The next step is to identify and analyze the strain.

[0076] 2. Identification of aflatoxin-removing strain ASAG57

[0077] According to the method described in the Bergey's Manual of Bacterial Identification (8th edition), strain ASAG57 was identified by morphological characteristics and physiological and biochemical characteristics. The specific results are as follows:

[0078] Morphological identification: Streak strain ASAG57 on PDA medium and culture at 30℃ for 48h. Observe the colony morphology, size, edge, surface protrusion shape, transparency, etc. Figure 2 As shown in the figure, the strain is white to milky white, bulging upwards, and has a smooth surface. After the strain is stained with methylene blue, the morphology of the strain is observed. The results are as follows Figure 3 As shown in Figure 2, strain ASAG57 is oval under an optical microscope. A scanning electron microscope was used to further observe the microscopic morphology of strain ASAG57. The results are shown in Figure 2. Figure 4 As shown, the cells are ellipsoidal in shape and measure 1.6–2.0 × 2.1–2.5 μm.

[0079] Molecular identification: 18S rDNA identification of strain ASAG57 was performed. Bacterial genomic DNA was extracted according to the instructions of the bacterial genomic DNA extraction kit as a template and 18S rDNA universal primers (18SF: 5'-GTAGTCATATGCTTGTCTC-3', 18SR: 5'-ATTCCCCGTTACCCGTTG-3') were used for PCR amplification to obtain its 18S rDNA. Among them, the PCR amplification system: template 1μL, Premix PrimeSTAR HS 12.5μL, 18S upstream and downstream primers (18SF and 18SR) 1μL each, double distilled water 10.5μL; PCR amplification reaction program: 98℃ pre-denaturation for 5min, 98℃ denaturation for 30s, 55℃ annealing for 20s, 72℃ extension for 1min, 30 cycles, 72℃ extension for 10min. The PCR product was entrusted to Shanghai Sangon Biotechnology Co., Ltd. for sequencing, and the sequencing results were compared in NCBI. The PCR product was detected by 1% agarose gel, excised, recovered, purified, and sequenced to obtain the nucleotide sequence of the strain's 18S rDNA as shown in SEQ ID NO. 1. After BLAST comparison with Genbank and further whole genome sequencing analysis, the strain ASAG57 was identified as Pichia guilliermondii. The phylogenetic tree was constructed using MEGE5.0 software ( Figure 5 ), the similarity between strain ASAG57 and M. guilliermondii MguEd003 was 99.8%.

[0080] The above identification results indicate that strain ASAG57 is Meyerozyma guilliermondii. Strain ASAG57 was deposited in the General Microbiological Center of China Microorganism Culture Collection Administration (referred to as: CGMCC, address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, China, Postal Code: 100101) on September 15, 2022, with a deposit number of CGMCC No. 25722 and a classification name of Meyerozyma guilliermondii.

[0081] Example 2 Aflatoxin removal properties of strain ASAG57

[0082] The strain ASAG57 was inoculated on YPD solid culture medium and cultured at 30°C for 48h. A single colony of strain ASAG57 was picked and cultured in 5mL YPD liquid culture medium at 30°C shaker overnight. It was transferred to 950mL YPD liquid culture medium with a 5% inoculation amount and cultured at 30°C 200r / min shaking for 48h to obtain fermentation broth. Take part of the fermentation broth for ultrasonic disruption to obtain a fermentation broth whole cell disruption liquid. Take part of the fermentation broth whole cell disruption liquid and heat it at 121°C for 20min to inactivate it, obtain an inactivated fermentation broth whole cell disruption liquid, and place it at 4°C for use. Take another part of the fermentation broth whole cell disruption liquid and treat it with a proteinase K reagent with a final concentration of 1mg / mL at 55°C for 1h to obtain a proteinase K-treated fermentation broth whole cell disruption liquid, which is placed at 4°C for use.

[0083] 5 mL of YPD liquid culture medium, fermentation broth, fermentation broth whole cell lysis liquid, inactivated fermentation broth whole cell lysis liquid, and fermentation broth whole cell lysis liquid treated with proteinase K were respectively placed in a toxin tube containing 50 μg AFB1. Three parallels were set for each reaction. After warming for 5 hours at 30°C and 200 r / min, 100 μL of sample was taken, 100 μL of methanol was added, and after mixing evenly, the supernatant was taken by centrifugation, and the residual amount (content) of AFB1 was detected by HPLC. The method was the same as in Example 1, wherein the AFB1 content detected by adding YPD liquid culture medium was used as the AFB1 content of the control group, and the AFB1 content detected by adding fermentation broth, fermentation broth whole cell lysis liquid, inactivated fermentation broth whole cell lysis liquid, and fermentation broth whole cell lysis liquid treated with proteinase K was used as the AFB1 content of the experimental group to calculate the AFB1 removal rate (%).

[0084] The results are as follows Figure 6 As shown in the figure, the AFB1 content in the control group remained basically unchanged, 95.55% of AFB1 could be removed from the fermentation broth within 5 hours, no AFB1 residue was detected in the fermentation broth whole cell lysate obtained by ultrasonic disruption of the fermentation broth, 80.33% of AFB1 could be removed from the inactivated fermentation broth whole cell lysate, and 70.85% of AFB1 could be removed from the fermentation broth whole cell lysate treated with proteinase K. The whole cell lysate of the fermentation broth had a significant AFB1 removal effect after high temperature inactivation and proteinase K treatment, respectively, indicating that this removal effect was not caused by the degradation of biological enzymes, but may be caused by the adsorption of substances such as cell wall polysaccharides and partial residues of denatured proteins, and high temperature would also promote the exposure of adsorbed residues of polysaccharides and denatured proteins to a certain extent, thus, the removal effect of high temperature inactivation was better than that of proteinase K treatment. In addition, the removal effect of the whole cell disrupted fermentation broth without high temperature inactivation is better than that after high temperature inactivation, indicating that the sample without high temperature inactivation has not only adsorption effect, but also catalytic conversion effect of biological enzyme, while high temperature inactivation causes the loss of biological enzyme activity, resulting in a decrease in the removal effect.

[0085] In summary, strain ASAG57 has efficient degradation and adsorption capacity for AFB1, that is, removal ability.

[0086] Example 3 Cloning, construction, expression and functional verification of aflatoxin B1 (AFB1) degrading enzyme gene

[0087] 1. Cloning of AFB1 degrading enzyme gene

[0088] Based on the whole genome information of strain ASAG57, aflatoxin degrading enzyme gene discovery was carried out. After sequence comparison and analysis, a new aflatoxin B1 (AFB1) degrading enzyme was screened and named MG2-4. Its nucleotide sequence is shown in SEQ ID NO.2, and its amino acid sequence is shown in SEQ ID NO.3. The upstream primer and downstream primer sequences of the gene are:

[0089] MG2-SacI-F:5'-C GAGCT CATGGCGAGACAATTTTATACAAAATC-3'(SEQ ID NO.4)

[0090] MG2-XhoI-R: 5'-CCG CTCGAG GTCAAACCCTGCAGCAAACTGC-3'(SEQ ID NO.5)

[0091] Restriction endonuclease Sac I and Xho I sites were added to the upstream and downstream primers, respectively (restriction endonucleases were underlined).

[0092] The fungal genomic DNA of strain ASAG57 was used as a template, and primers MG2-SacI-F and MG2-XhoI-R were used for PCR amplification to obtain the PCR product containing the AFB1 degradation enzyme gene MG2-4. The PCR amplification system: template 1μL, Premix PrimeSTAR HS 25μL, primers MG2-SacI-F and MG2-XhoI-R 1μL each, double distilled water 22μL; PCR reaction program: 98℃ pre-denaturation for 5min, 98℃ denaturation for 30s, 55℃ annealing for 30s, 72℃ extension for 1min, 30 cycles, 72℃ extension for 10min.

[0093] 2. Construction and Expression

[0094] 1. Build

[0095] The PCR product containing the AFB1 degrading enzyme gene MG2-4 and the Escherichia coli expression vector pET-28a(+) were double-digested with Sac I and Xho I, respectively. Among them, the enzyme digestion system of the PCR product containing the AFB1 degrading enzyme gene MG2-4 was 5μL of the PCR product containing the AFB1 degrading enzyme gene MG2-4, 3μL of 10×Buffer, 1μL of Sac I, 1μL of Xho I, and 20μL of double distilled water. The pET-28a(+) enzyme digestion system was pET-28a(+) 16μL, 2μL of 10×Buffer, 1μL of Sac I, and 1μL of Xho I. The enzyme digestion was performed at 37°C for 1h. The agarose gel electrophoresis results of the double-enzyme digestion products of the PCR product containing the AFB1 degrading enzyme gene MG2-4 and the Escherichia coli expression vector pET-28a(+) are shown in Figure 7 As shown. The PCR product containing the AFB1 degrading enzyme gene MG2-4 and the double-enzyme digestion product of the Escherichia coli expression vector pET-28a(+) were recovered by agarose gel electrophoresis and connected to obtain a connection product. The connection system and conditions are as follows: 1.5 μL of the double-enzyme digestion product of the Escherichia coli expression vector pET-28a(+) was added to 6 μL of the double-enzyme digestion product of the PCR product containing the AFB1 degrading enzyme gene MG2-4, heated at 62°C for 2 minutes, and immediately placed on ice, and 7.5 μL of Solution I was added thereto, and the connection was carried out at 16°C overnight.

[0096] The ligation product was transformed into E. coli DH5α competent cells. After plating and overnight culture, a single colony was picked for PCR amplification verification. The results were as follows: Figure 8 As shown, the size of the target fragment is consistent with the size of the AFB1 degradation enzyme gene MG2-4. Further sequencing verification was performed, and plasmids were extracted from the correct transformants verified by sequencing to obtain the recombinant plasmid pET-28a(+)-MG2-4 of the AFB1 degradation enzyme gene (the plasmid map is shown in Fig. 9 The recombinant plasmid pET-28a(+)-MG2-4 was transferred into the Escherichia coli expression host Rosetta (DE3) to obtain the recombinant strain Rosetta [pET-28a(+)-MG2-4].

[0097] 2. Expression

[0098] The recombinant strain Rosetta[pET-28a(+)-MG2-4] was inoculated into 5 mL of LB medium containing 50 μg / mL kanamycin and shaken at 37°C overnight. A 1% inoculum was transferred to 100 mL of LB medium containing 50 μg / mL kanamycin and cultured at 37°C and 200 r / min. OD 600When it reached 0.6, the inducer IPTG (final concentration 0.8mM) was added and induced at 18℃ for 18h. The cells were collected by centrifugation at 7000r / min for 10min at 4℃, resuspended in 40mL of PBS, ultrasonically broken under ice bath conditions, and centrifuged at 12000r / min for 20min at 4℃. The supernatant was collected to obtain the crude enzyme solution of AFB1 degrading enzyme MG2-4, and SDS-PAGE was performed to verify the protein expression. The theoretical molecular size of AFB1 degrading enzyme MG2-4 is 35.7kDa, and the theoretical isoelectric point is 5.6.

[0099] 3. Purification

[0100] The crude enzyme solution was purified using a Ni column, and PBS was used as a balance solution. After collecting the sample penetration solution, it was eluted using imidazole solutions from low to high concentrations in sequence, and each concentration of imidazole elution sample was collected. The above-collected imidazole elution samples and bovine serum albumin samples of known concentrations were used for SDA-PAGE electrophoresis. The results of SDA-PAGE electrophoresis showed that the supernatant of bacterial cell crushing contained the target protein. The purified enzyme solution of AFB1 degrading enzyme MG2-4 was eluted under the condition of 500mM imidazole concentration (such as Fig.10 shown).

[0101] 3. Functional Verification

[0102] 100 μL PBS, Rosetta (DE3) disruption solution containing pET-28a (+) empty plasmid, and purified enzyme solution of AFB1 degrading enzyme MG2-4 were added to 500 μL (AFB1 final concentration 2 μg / mL) reaction system, respectively. After reacting at 30° C. for 1 h, 500 μL methanol was added to terminate the reaction. The supernatant was collected by centrifugation, and the residual AFB1 amount (content) was detected by HPLC. The method was the same as that in Example 1. The AFB1 content detected by adding PBS was used as the AFB1 content of the control group, and the AFB1 content detected by adding Rosetta (DE3) disruption solution containing pET-28a (+) empty plasmid and purified enzyme solution of AFB1 degrading enzyme MG2-4 were used as the AFB1 content of the experimental group to calculate the AFB1 degradation rate (%).

[0103] Wherein, AFB1 degradation rate (%) = (AFB1 content of control group - AFB1 content of experimental group) / AFB1 content of control group × 100.

[0104] The results showed that compared with the control group supplemented with PBS, the Rosetta (DE3) fragmentation solution containing pET-28a (+) empty plasmid had no degradation activity on AFB1, and the purified enzyme solution of AFB1 degrading enzyme MG2-4 had a degradation rate of up to 89.6% on AFB1.

[0105] Example 4 Construction, expression and effect verification of AFB1 degrading enzyme MG2-4 in Pichia pastoris

[0106] 1. Cloning of AFB1 degrading enzyme gene

[0107] Using the fungal genomic DNA of strain ASAG57 as a template, primers MG2-SnaB IF and MG2-Not IR were used to perform PCR amplification to obtain a PCR product containing the AFB1 degradation enzyme gene MG2-4. The PCR amplification system and PCR reaction procedure were the same as those in Example 3.

[0108] The primer sequences are as follows:

[0109] MG2-SnaB IF:

[0110] 5'-CGC TACGTA ATGGCGAGACAATTTTATACAAAATC-3'(SEQ ID NO.6)

[0111] MG2-Not IR:

[0112] 5'-ATAAGAAT GCGGCCGC GTCAAACCCTGCAGCAAACTGC-3'(SEQ ID NO.7)

[0113] Restriction endonuclease SnaB I and Not I cleavage sites were added to the upstream and downstream primers, respectively (restriction endonucleases were underlined).

[0114] The PCR product containing the AFB1 degradation enzyme gene MG2-4 was double-digested with restriction endonucleases SnaB I and Not I. The 100 μL digestion system was as follows: 40 μL of the PCR product containing the AFB1 degradation enzyme gene MG2-4, 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 hours, the MG2-4 fragment double-digested with SnaB I and Not I was recovered by agarose gel electrophoresis.

[0115] 2. Construction and Expression

[0116] 1. Build

[0117] The expression vector pPIC9K was double-digested with restriction endonucleases SnaB I and Not I. The digestion system for 100 μL was as follows: vector pPIC9K 20 μL, 10×H buffer 10 μL, SnaB I 5 μL, Not I 5 μL, ddH2O 65 μL. After digestion at 37°C for 4 h, the expression vector pPIC9K double-digested with SnaB I and Not I was recovered by agarose gel electrophoresis.

[0118] The MG2-4 fragment double-digested with SnaB I and Not I was connected to the expression vector pPIC9K double-digested with SnaB I and Not I to construct the recombinant yeast expression plasmid pPIC9K-MG2-4. The connection system is as follows: 4.5 μL of the expression vector pPIC9K double-digested product, 3 μL of the MG2-4 fragment double-digested with SnaB I and Not I, and 7.5 μL of Solution I. The connection was carried out overnight at 16°C, and the connection product was transformed into Escherichia coli DH5α, and the transformant was picked for sequencing verification. The plasmid extracted from the transformant verified by sequencing is the recombinant yeast expression plasmid pPIC9K+MG2-4 (the plasmid map is shown in Fig.11 shown).

[0119] 2. Expression

[0120] The recombinant yeast expression plasmid pPIC9K+MG2-4 was linearized with Sal I. After the linearization product was purified, it was transformed into Pichia GS115 by electroporation and coated with MD solid medium. The colonies grown on the MD plate were Pichia engineering strains, that is, the recombinant Pichia strain GS115 [pPIC9K+MG2-4] was obtained, and then multi-copy transformants were screened on YPDYPD solid medium containing different concentrations of geneticin G418. A single multi-copy transformant was picked and inoculated into BMGY medium, and after shaking culture at 30°C and 200r / min for 24 hours, it was transferred to BMMY medium, shaken and cultured at 30°C and 200r / min, and 0.5% methanol was added every 24 hours. After induced expression for 4 days, the supernatant was removed by centrifugation to obtain the crude enzyme solution of the recombinant AFB1 degrading enzyme MG2-4.

[0121] 3. Effect Verification

[0122] The crude enzyme solution of recombinant AFB1 degrading enzyme MG2-4 was used for in vitro AFB1 degradation experiments. The degradation reaction system contained 250 μL crude enzyme solution, 10 μL NADPH solution and 1 μg AFB1, and then the total volume of the system was supplemented to 500 μL with PBS. This treatment was set as the enzyme solution degradation group. The inactivated enzyme solution degradation group used crude enzyme solution heated at 121°C for 20 min instead of crude enzyme solution. The control group used BMMY liquid culture medium instead of crude enzyme solution. Three parallels were set for each group. All three groups were placed in a shaker at 30°C and reacted at 200 r / min. After 1 hour, 500 μL of methanol was added to the degradation reaction system to terminate the reaction. The supernatant was centrifuged and the residual AFB1 amount (content) was detected by HPLC. The method was the same as that in Example 3. The AFB1 content detected in the control group with added BMMY liquid culture medium was used as the AFB1 content of the control group, and the AFB1 content detected in the enzyme solution degradation group with added crude enzyme solution of recombinant AFB1 degrading enzyme MG2-4 and the inactivated enzyme solution degradation group with crude enzyme solution heated at 121°C for 20 min were used as the AFB1 content of the experimental group to calculate the AFB1 degradation rate (%).

[0123] The results are as follows Fig.12 As shown, the crude enzyme solution of the recombinant AFB1 degrading enzyme MG2-4 in the enzyme solution degradation group can degrade 95.28% of AFB1 within 1 hour, and there is no obvious change in the AFB1 content of the control group and the inactivated enzyme solution degradation group, indicating that the recombinant AFB1 degrading enzyme MG2-4 has a higher activity in degrading AFB1.

[0124] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not limitations on the implementation methods of the present invention. For ordinary technicians in the relevant field, other different forms of changes or modifications can be made based on the above description. It is impossible to list all the implementation methods here. All obvious changes or modifications derived from the technical solution of the present invention are still within the protection scope of the present invention.

Claims

1. A strain of Pichia guilliermong ( Meyerozyma guilliermondii ), characterized in that, The preservation number of the Pichia guilliermong is CGMCC No.25722.

2. A microbial preparation, characterized in that: The active ingredients of the microbial preparation include the Pichia guilliermondii yeast according to claim 1 or its fermentation broth or its fermentation broth whole cell fragmentation liquid or its inactivated fermentation broth whole cell fragmentation liquid or its proteinase K treated fermentation broth whole cell fragmentation liquid or its culture.

3. Use of the Pichia guilliermongins described in claim 1 or the microbial preparation described in claim 2 in any of the following: B1) Application in removing aflatoxins from samples; B2) Application in the preparation of aflatoxin-free products.

4. Use of an aflatoxin degrading enzyme or a gene encoding the aflatoxin degrading enzyme or an expression cassette, a recombinant vector or a recombinant microorganism containing the gene of the aflatoxin degrading enzyme in any of the following: B1) Application in removing aflatoxins from samples; B2) Application in the preparation of products for removing aflatoxin from samples; in, The amino acid sequence of the aflatoxin degrading enzyme is shown in A1) or A2): A1) The amino acid sequence of the protein is shown in SEQ ID No. 3; 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. 3; The nucleotide sequence of the aflatoxin degrading enzyme gene is shown in SEQ ID No.

2.

5. A method for removing aflatoxin, characterized in that: The steps include: Adding the Pichia guilliermong described in claim 1 or the microbial preparation described in claim 2 to a sample containing aflatoxin, and culturing the sample to remove the aflatoxin from the sample; or, Adding aflatoxin degrading enzyme to a sample containing aflatoxin to react and remove aflatoxin from the sample; Wherein, the amino acid sequence of the aflatoxin degrading enzyme is shown as A1) or A2): A1) The amino acid sequence of the protein is shown in SEQ ID No. 3; 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.

3.

6. The method according to claim 5, characterized in that The culture conditions are 25-35°C, 50-300 r / min, and a warm bath for 0.5-12 hours; The reaction conditions are 25-35° C. and 50-300 r / min for 0.5-12 hours.

7. The use according to claim 3 or 4 or the method according to claim 5, characterized in that: The sample is grain and its processing by-products, feed or food.

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