CN113355304B - Protein CpoC with zearalenone degrading enzyme activity and gene and application thereof - Google Patents

Abstract

The present disclosure relates to a protein having zearalenone degrading enzyme activity, a gene encoding the protein, a recombinant vector into which the gene is inserted, a transformant transformed with the gene, a method for preparing zearalenone degrading enzyme, and an application of zearalenone degrading enzyme in treating agricultural products. The application of the novel zearalenone degrading enzyme is greatly facilitated, and the requirements of the zearalenone degrading enzyme in practical application are better met.

Description

Protein CpoC with zearalenone degrading enzyme activity and gene and application thereof

Technical Field

The present disclosure relates to the technical field of genetic engineering, and in particular, to a protein having zearalenone degrading enzyme activity, a gene encoding the protein, a recombinant vector into which the gene is inserted, a transformant transformed with the gene, a method for preparing zearalenone degrading enzyme, and an application of zearalenone degrading enzyme in agricultural product treatment.

Background

Mycotoxins are toxic metabolites or secondary metabolites produced by fungi, and the high toxicity and strong carcinogenicity of the mycotoxins seriously threaten the production performance of animals and the health of human beings and bring huge loss to the food industry, the feed industry and the animal husbandry. The detoxification method of mycotoxin mainly comprises physical, chemical and biological detoxification methods, and the biological detoxification method can degrade mycotoxin into nontoxic substances and has the advantages of strong specificity, high conversion efficiency, safety, environmental protection and the like, but so far, high-efficiency broad-spectrum mycotoxin degrading enzymes capable of being applied industrially are still few.

Zearalenone (ZEN), which is a secondary metabolite of estrogenic-like activity, is one of the most harmful mycotoxins. Currently, research on ZEN degrading enzymes mainly focuses on fungus-derived lactone hydrolase ZHD101 and homologues thereof, the degrading enzymes can effectively convert ZEN into nontoxic products, but bottleneck problems to be solved, such as low degradation activity of intermediate product alpha-zearalenol, poor thermal stability, low heterologous expression level and the like, exist, and therefore more enzyme resources for degrading zearalenone need to be explored and mined.

Disclosure of Invention

In order to further satisfy the requirements of practical applications, the present disclosure provides a protein having zearalenone degrading enzyme activity, a gene encoding the protein, a recombinant vector into which the gene is inserted, a transformant transformed with the gene, a method for preparing zearalenone degrading enzyme, and use of zearalenone degrading enzyme in treating agricultural products.

The present disclosure provides, in a first aspect, a protein having zearalenone degrading enzymatic activity, which is the following protein (a) or (b): (a) a protein consisting of an amino acid sequence shown in SEQ ID No. 2; (b) and (b) the protein derived from the (a) and having zearalenone degrading enzyme activity, wherein the amino acids in the (a) are substituted, deleted or added with n amino acids, and n is any integer between 1 and 4.

In a second aspect of the present disclosure, there is provided a gene encoding the following protein (a) or (b): (a) a protein consisting of an amino acid sequence shown in SEQ ID No. 2; (b) and (b) the protein which is derived from the protein (a) and has zearalenone degrading enzyme activity, wherein n is any integer between 1 and 4, and the protein is obtained by substituting, deleting or adding n amino acids in the amino acid sequence in the protein (a).

In a third aspect of the present disclosure, there is provided a recombinant vector, wherein the recombinant vector comprises the gene of the second aspect.

The fourth aspect of the present disclosure provides a transformant, wherein a host cell of the transformant is a genetically engineered bacterium; the gene introduced into the transformant includes the gene of the second aspect, or the recombinant vector introduced into the transformant is the recombinant vector of the third aspect.

The fifth aspect of the present disclosure provides a method for preparing zearalenone degrading enzyme, wherein the degrading enzyme method comprises inoculating the transformant of the fourth aspect into a culture medium for culturing.

In a sixth aspect, the present disclosure provides a zearalenone degrading enzyme for treating an agricultural product, wherein the zearalenone degrading enzyme comprises the protein of the first aspect.

By the technical scheme, the protein with the activity of the zearalenone degrading enzyme, the gene for coding the protein, the recombinant vector inserted with the gene, the transformant transformed with the gene, the method for preparing the zearalenone degrading enzyme and the application of the zearalenone degrading enzyme in treating agricultural products are provided, the foundation is laid for producing the novel zearalenone degrading enzyme with high yield at low cost, and the zearalenone degrading enzyme has important application potential in biodegradation of mycotoxin in the field of agricultural products.

Additional features and advantages of the disclosure will be set forth in the detailed description which follows.

Drawings

The accompanying drawings, which are included to provide a further understanding of the disclosure and are incorporated in and constitute a part of this specification, illustrate embodiments of the disclosure and together with the description serve to explain the disclosure without limiting the disclosure. In the drawings:

FIG. 1 is a gel diagram of the purification of protein CpoC. Lane 1, crude enzyme solution of uninduced recombinant bacteria; lane 2, crude enzyme solution of recombinant bacteria after induction; lane 3, NTA-10 eluate; lane 4, NTA-20 eluate; lane 5, NTA-50 eluate; lane 6, NTA-100 eluate; lane 7, NTA-150 eluate; lane 8, NTA-200 eluate.

FIG. 2 is an HPLC chromatogram of protein CpoC for degradation of zearalenone.

FIG. 3 is an alignment analysis of the sequences of the degrading enzymes CpoC and ZHD 101.

Detailed Description

The following detailed description of specific embodiments of the present disclosure is provided in connection with the accompanying drawings. It should be understood that the detailed description and specific examples, while indicating the present disclosure, are given by way of illustration and explanation only, not limitation.

The first aspect of the present disclosure provides a protein having zearalenone degrading enzyme activity, wherein the protein is the following protein (a) or (b):

(a) a protein consisting of an amino acid sequence shown in SEQ ID No. 2;

(b) and (b) the protein derived from the (a) and having zearalenone degrading enzyme activity, wherein the amino acids in the (a) are substituted, deleted or added with n amino acids, and n is any integer between 1 and 4.

The protein with zearalenone degrading enzyme activity provided by the first aspect of the disclosure is the protein CpoC. Wherein n may be 1, 2, 3 or 4.

The activity of zearalenone degrading enzyme can be measured with reference to DB42/T1630-2021 (measurement of zearalenone degrading enzyme activity). The zearalenone degrading enzyme activity unit may be defined as including: the amount of enzyme required to degrade 1. mu.g of zearalenone from a zearalenone solution having a concentration of 10. mu.g/mL per minute at 37 ℃ and a pH of 5.5 was one zearalenone degrading enzyme activity unit (U).

In a second aspect of the present disclosure, there is provided a gene encoding the following protein (a) or (b):

(a) a protein consisting of an amino acid sequence shown in SEQ ID No. 2;

(b) and (b) the protein which is derived from the protein (a) and has zearalenone degrading enzyme activity, wherein n is any integer between 1 and 4, and the protein is obtained by substituting, deleting or adding n amino acids in the amino acid sequence in the protein (a).

According to the disclosure, the gene is a DNA molecule having a nucleotide sequence shown in SEQ ID NO. 1.

In a third aspect of the present disclosure, there is provided a recombinant vector, wherein the recombinant vector comprises the gene of the second aspect.

According to the disclosure, the recombinant vector is a recombinant expression vector, and the recombinant expression vector is a DNA molecule with a nucleotide sequence shown in SEQ ID NO. 3.

The fourth aspect of the present disclosure provides a transformant, wherein a host cell of the transformant is a genetically engineered bacterium; the gene introduced into the transformant includes the gene of the second aspect, or the recombinant vector introduced into the transformant is the recombinant vector of the third aspect.

According to the disclosure, the genetically engineered bacterium is one of escherichia coli, bacillus subtilis, pichia pastoris and saccharomyces cerevisiae.

The fifth aspect of the present disclosure provides a method for preparing a zearalenone degrading enzyme, wherein the degrading enzyme method comprises inoculating the transformant of the fourth aspect into a culture medium for culturing. Among them, the medium and the culture conditions may be selected as appropriate in a known manner. The cultured material contains the protein having zearalenone degrading enzyme provided by the first aspect of the present disclosure, and thus has zearalenone degrading enzyme activity, and can be directly used as a zearalenone degrading enzyme composition as required, or the protein having zearalenone degrading enzyme activity provided by the first aspect of the present disclosure can be purified as required and then used.

In a sixth aspect, the present disclosure provides a zearalenone degrading enzyme for treating an agricultural product, wherein the zearalenone degrading enzyme comprises the protein of the first aspect.

According to the present disclosure, wherein the produce comprises at least one of grain, nuts, fruit; preferably, the grain comprises corn and the nuts comprise peanuts.

The present disclosure is further illustrated by the following examples, but is not to be construed as being limited thereby.

Example 1

This example constructs transformants expressing the CpoC gene.

The expression plasmid pET28a is commercially available from Merck, Germany; coli BL21(DE3) is a commercially available product from Beijing Novowed company.

Firstly, amplifying a metagenome of a moldy corn, straw and rhizome sample by using the following primers to obtain a CpoC gene: CpoC-F: 5'-GGATCCATGAGCAGCGGCTTCTTCAC-3' (SEQ ID NO.4), CpoC-R: 5'-CTCGAGTCTACCCTTCGATGAAGGCC-3' (SEQ ID NO. 5); the PCR reaction procedure is shown in Table 1; after the PCR product is recovered by glue, connecting the PCR product to a pET-28a vector containing a sticky end obtained by BamHI/XhoI double enzyme digestion through a recombinase to construct an escherichia coli recombinant expression vector pET-CpoC; the expression vector is transformed into escherichia coli BL21(DE3), the inserted sequence is verified to be correct through PCR, enzyme digestion and sequencing, a transformant is obtained, and the transformant is named as BL 21-pET-CpoC.

TABLE 1 PCR reaction procedure

Example 2

This example was used to prepare pure protein.

Inducible expression of the degrading enzyme CpoC: (1) inoculating the transformant to 20mL of LB liquid medium added with antibiotics in an inoculation amount of 1%, and culturing overnight in a shaker at 37 ℃; (2) the following day by OD₆₀₀Transferring the bacterial liquid into 500mL of LB liquid culture medium added with kanamycin for the inoculation amount of 0.1 initial concentration, and culturing at 37 ℃ until the bacterial liquid concentration is 0.6-0.8; (3) IPTG (final concentration 0.5mmol/L) is added for protein induction expression, and the induction condition is 16 ℃ and 20 h.

Affinity chromatography purification of recombinant degrading enzyme CpoC: (1) centrifuging the induced bacterial liquid, collecting thalli at 5000rpm for 10min, and resuspending the thalli by NTA-0; (2) ultrasonic crushing of bacterial liquid: placing a centrifugal tube containing suspended bacteria in a beaker of an ice-water mixture, placing the centrifugal tube in an ultrasonication instrument for ultrasonication for 5-10 min, wherein the ultrasonication instrument has the following setting procedures: stopping ultrasound for 3s and 5s, wherein the power is less than 400W; (3) 13000Xg of the sample after ultrasonic crushing is centrifuged for 30min, the supernatant and the precipitate of the crushing liquid are respectively collected by a centrifuge tube, and the crushing supernatant obtained by centrifugation is the crude enzyme liquid. (4) Taking out the nickel column, after the ethanol flows out, firstly washing the nickel column twice by using deionized water, and then balancing the column by using NTA-0, wherein the flow rate is kept at 1 mL/min; hanging the crude enzyme solution on a column, penetrating twice, and keeping the flow rate the same as the above; (5) gradient eluting with prepared NTA-10, NTA-20, NTA-50, NTA-100, NTA-150, NTA-200, and NTA-300, detecting protein with protein detection solution, and collecting eluate; washing the nickel column with 20% ethanol solution, and storing the nickel column in a refrigerator at 4 deg.C; and (3) replacing the buffer solution of the protein solution by ultrafiltration centrifugation to remove imidazole in the protein solution, wherein the obtained protein solution is the enzyme solution.

In the above experiment: IPTG is an inducer inducing recombinant strains to express the degrading enzyme, CpoC protein, and is not a substrate for the catalytic reaction.

The experimental results of this example show that: the CpoC gene is capable of large soluble expression in E.coli. Elution with imidazole eluents of 50mM, 100mM and 150mM showed a single protein band at approximately 35kDa, which is closer to the predicted size of the CpoC fused His-Tag, indicating that CpoC was eluted in buffers containing 50-150mM imidazole. The SDS-PAGE electrophoresis of the purified CpoC protein has clear background, no other protein bands, higher protein purity and larger expression amount, as shown in FIG. 2. The purified protein was subjected to desalting column to displace the buffer and remove imidazole for further study of enzymatic properties.

Example 3

This example was used to determine the degradation activity of the degrading enzyme CpoC.

Experimental materials: degrading the enzyme to obtain the CpoC protein: purified CpoC protein from example 2; zearalenone (ZEN): chromatographic grade, purchased from Shanghai-derived leaf Biotech, Inc.

Determination of the degradation activity of recombinase CpoC: (1) preparing ZEN standard working mother liquor: 5mL of methanol was added to 25mg of ZEN standard by syringe, and mixed by shaking to give a final concentration of 5 mg/mL. Sealing and storing, and standing at-20 deg.C for use. (2) Preparing a reaction mixed system: mu.l of enzyme solution with 0.05mol-L Tris-HCl buffer (pH 7.0) as buffer and 2. mu.l ZEN standard stock solution (5mg/mL) were added to give a final concentration of 20. mu.g/mL. (3) The reaction mixture was incubated at 37 ℃ for various times (30min, 1h, 2h, 8 h). After incubation, the reaction was stopped by adding an equal volume of methanol, and then the ZEN concentration in the reaction enzyme solution was quantified by an Agilent HPLC system. The detection conditions of the high performance liquid chromatography are as follows: a chromatographic column: agilent C18 chromatography column, 4.6mm × 250mm × 5 μm; mobile phase: acetonitrile-water (containing 0.1% formic acid) (10: 90); flow rate: 0.8 mL/min; column temperature: 25 ℃; detection wavelength: λ 274 nm; sample introduction amount: 10 μ L. ZEN degradation rate (%) [20(μ g/mL) -remaining ZEN concentration (μ g/mL) ]/20 × 100; results represent the average of triplicates; the enzyme activity unit (U) is defined as the amount of enzyme capable of converting 1 microgram of ZEN in 1 hour at 37 ℃ under the reaction condition of 4 hours.

Calculating enzyme activity (enzyme activity unit contained in each milliliter of enzyme solution):

wherein, the enzyme activity is defined as an enzyme activity unit (U) contained in each mL of enzyme solution, and the enzyme activity is expressed in the form of average value +/-standard deviation. The dilution factor is the dilution factor of the enzyme solution to be detected in the enzyme activity reaction system, and 20 is the unit conversion of the volume of the enzyme solution.

The experimental results of this example show that: CpoC was incubated with 20. mu.g/mL ZEN under the same conditions and sampled for HPLC detection at 30min, 1h, 2h, 3h, 4h and 5h respectively, and the HPLC profile of degradation of ZEN by degrading enzyme CpoC as a function of time is shown in FIG. 2. The change of the ZEN degradation rate with time is shown in Table 1, the degradation rate of ZEN reaches 41% after 1 hour of CpoC incubation, the degradation rate of ZEN reaches 98% after 3 hours of CpoC incubation and almost completely degrades, the residual ZEN amount of 3-5 hours of CpoC incubation is almost unchanged, the ZEN degradation rate is stabilized to be nearly 100%, the specific enzyme activity is 960.92 +/-8.30U/mL, and the degradation rate of ZEN reaches 100% by CpoC. Meanwhile, the sequence alignment analysis shows that the degrading enzyme CpoC has only 25.51 percent of similarity with the currently reported zearalenone degrading enzyme ZHD101 (figure 3), and the CpoC is a novel zearalenone hydrolase.

TABLE 1 degradation rate of ZEN at different CpoC times

Time	Rate of degradation
		30min	22.21％±0.29％
1h	41.68％±1.52％
		2h	75.74％±2.13％
3h	98.00％±0.78％
		4h	99.17％±0.81％
5h	99.16％±0.10％

The method enriches and purifies efficient and stable zearalenone degrading microorganisms from moldy corn, straw and rhizome samples, utilizes a metagenome sequencing method to excavate a zearalenone degrading enzyme gene, discovers a novel efficient zearalenone degrading enzyme CpoC for the first time, and has important application potential in biodegradation of mycotoxin zearalenone in the fields of feed, food, livestock raising and the like.

The preferred embodiments of the present disclosure are described in detail with reference to the accompanying drawings, however, the present disclosure is not limited to the specific details of the above embodiments, and various simple modifications may be made to the technical solution of the present disclosure within the technical idea of the present disclosure, and these simple modifications all belong to the protection scope of the present disclosure.

It should be noted that, in the foregoing embodiments, various features described in the above embodiments may be combined in any suitable manner, and in order to avoid unnecessary repetition, various combinations that are possible in the present disclosure are not described again.

In addition, any combination of various embodiments of the present disclosure may be made, and the same should be considered as the disclosure of the present disclosure, as long as it does not depart from the spirit of the present disclosure.

Sequence listing

<110> institute of biotechnology of Chinese academy of agricultural sciences

<120> protein CpoC with zearalenone degrading enzyme activity, gene and application thereof

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gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat aatactgttg atgggtgtct 4140

ggtcagagac atcaagaaat aacgccggaa cattagtgca ggcagcttcc acagcaatgg 4200

catcctggtc atccagcgga tagttaatga tcagcccact gacgcgttgc gcgagaagat 4260

tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc taccatcgac accaccacgc 4320

tggcacccag ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca 4380

gggccagact ggaggtggca acgccaatca gcaacgactg tttgcccgcc agttgttgtg 4440

ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc ttccactttt tcccgcgttt 4500

tcgcagaaac gtggctggcc tggttcacca cgcgggaaac ggtctgataa gagacaccgg 4560

catactctgc gacatcgtat aacgttactg gtttcacatt caccaccctg aattgactct 4620

cttccgggcg ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg gtgtccggga 4680

tctcgacgct ctcccttatg cgactcctgc attaggaagc agcccagtag taggttgagg 4740

ccgttgagca ccgccgccgc aaggaatggt gcatgcaagg agatggcgcc caacagtccc 4800

ccggccacgg ggcctgccac catacccacg ccgaaacaag cgctcatgag cccgaagtgg 4860

cgagcccgat cttccccatc ggtgatgtcg gcgatatagg cgccagcaac cgcacctgtg 4920

gcgccggtga tgccggccac gatgcgtccg gcgtagagga tcgagatctc gatcccgcga 4980

aattaatacg actcactata ggggaattgt gagcggataa caattcccct ctagaaataa 5040

ttttgtttaa ctttaagaag gagatatacc atgggcagca gccatcatca tcatcatcac 5100

agcagcggcc tggtgccgcg cggcagccat atggctagca tgactggtgg acagcaaatg 5160

ggtcgcggat ccatgagcag cggcttcttc accaccggcg acggcgcgca gatcttcttc 5220

aaggactggg gtcccaagga cgcgcagccg gttgtcttcc accacggctg gcccctgtcg 5280

tcggacgact gggacgccca gatgatgttc ttcctgctga aaggctaccg cgtcatcgcc 5340

catgaccgtc gcggtcacgg ccgatccagc cagaccgaca ccggcaacga gatggacacc 5400

tacgccgccg acgtcatcgc tttggcgcgc catctggatc tgaagaatgc ggtccacatc 5460

ggccactcga ccggcggcgg cgaagtcgcc cgctacgtgg cccgcgccga gcccggccgc 5520

gtcgccaagg ctgtgctggt cggggccgtg ccgccgatca tgctgaagac cgcggccaat 5580

ccgggcggcc tgccgatgga ggtgttcgac ggcttccgcg cggcgctgct gtccaaccgc 5640

gcccagttct tccacgacgt ggcggcgggc ccgttctacg gcttcaaccg cgacggcgcg 5700

aaggtctcgc cggccacggt cgagaactgg tggcgccagg gcatgatggg cggcgccaag 5760

gcccactacg actgcatcac cgccttctcg gagaccgact tcaccgagga tttgaaggcg 5820

atcgacgtcc cggtgctgat cctgcacggc gaggacgatc agatcgtgcc gatcgccgac 5880

agcgccgagc tggccatcaa gctggtcaag aacggcaagt tgatcaccta tcccggcttc 5940

ccgcacggca tggccaccac cgaggccgag acgatcaaca aggacctgct ggccttcatc 6000

gaagggtaga ctcgagcacc accaccacca ccactgagat ccggctgcta acaaagcccg 6060

aaaggaagct gagttggctg ctgccaccgc tgagcaataa ctagcataac cccttggggc 6120

ctctaaacgg gtcttgaggg gttttttgct gaaaggagga actatatccg gat 6173

<210> 4

<211> 26

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

ggatccatga gcagcggctt cttcac 26

<210> 5

<211> 26

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

ctcgagtcta cccttcgatg aaggcc 26

Claims (11)

1. A protein having zearalenone degrading enzyme activity, which is characterized by being a protein (a) as follows:

(a) a protein consisting of an amino acid sequence shown in SEQ ID NO. 2.

2. A gene encoding the following protein (a):

(a) a protein consisting of an amino acid sequence shown in SEQ ID NO. 2.

3. The gene according to claim 2, wherein the gene is a DNA molecule having a nucleotide sequence represented by SEQ ID No. 1.

4. A recombinant vector comprising the gene of claim 2 or 3.

5. The recombinant vector according to claim 4, wherein the recombinant vector is a recombinant expression vector, and the recombinant expression vector is a DNA molecule with a nucleotide sequence shown in SEQ ID NO. 3.

6. A transformant, wherein a host cell of the transformant is a genetically engineered bacterium; the gene introduced into the transformant includes the gene of claim 2 or 3, or the recombinant vector introduced into the transformant is the recombinant vector of claim 4 or 5.

7. The transformant according to claim 6, wherein the starting strain of the genetically engineered bacterium is one of Escherichia coli, Bacillus subtilis, Pichia pastoris and Saccharomyces cerevisiae.

8. A method for producing a zearalenone degrading enzyme, wherein the method comprises inoculating the transformant according to claim 6 or 7 into a culture medium and culturing.

9. Use of a zearalenone degrading enzyme for treating an agricultural product, wherein the zearalenone degrading enzyme is the protein of claim 1.

10. The use of claim 9, wherein the produce comprises at least one of grain, nuts, fruit.

11. The use of claim 10, wherein the grain comprises corn and the nuts comprise peanuts.

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