CN107099520B

CN107099520B - Zearalenone degrading enzyme and coding gene and application thereof

Info

Publication number: CN107099520B
Application number: CN201710516347.5A
Authority: CN
Inventors: 张桂敏; 王美星; 尹李峰; 周玉玲; 马延和
Original assignee: Hubei University
Current assignee: Hubei University
Priority date: 2017-06-29
Filing date: 2017-06-29
Publication date: 2020-10-16
Anticipated expiration: 2037-06-29
Also published as: CN107099520A

Abstract

The invention relates to a zearalenone degrading enzyme and a coding gene thereof, wherein the degrading enzyme has an amino acid sequence shown in SEQ ID No.1, or is a conservative variant obtained by deletion, substitution, insertion or/and addition of conservative mutation of one to several amino acids on the basis of the amino acid sequence shown in SEQ ID No. 1. The zearalenone degrading enzyme disclosed by the invention has the advantages of high enzyme activity, good pH tolerance and the like, can be widely applied to enzymolysis of zearalenone and several derivatives thereof, and is particularly good in substrate specificity of zearalenone and beta-zearalenol.

Description

Zearalenone degrading enzyme and coding gene and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a zearalenone degrading enzyme, and a coding gene and application thereof.

Background

Zearalenone, which was first isolated from corn, is a nonsteroidal estrogen mycotoxin that can be produced by many fusarium species and is produced by the crop before and after harvest. Zearalenone is always found in many crops and grain by-products including corn, barley, wheat, and the like, especially in environments suitable for fungal growth.

There are many derivatives of zearalenone, such as zearalenol, which enter the food chain through contaminated crops and accumulate in the human and animal body, causing damage to the organism. Zearalenone and its derivatives have a chemical structure similar to natural estrogens, so they can competitively bind to estrogen receptors, causing external and internal genital changes and reproductive disorders, leading to hyperestrogenia and infertility, and such toxins can also stimulate the growth of breast cancer cell lines and cause carcinogenesis in mice.

In view of the hazards of such toxins, zearalenone and the like must be present in cereals, food and feed in amounts below certain standards. Since zearalenone and the like are extremely stable, removal of such toxins using traditional physical and chemical methods is inefficient. To address these problems, one promising strategy to reduce such toxin contamination is enzymatic degradation. The enzyme degradation not only can efficiently convert toxin into a non-toxic product, is safe and environment-friendly, but also has strong specificity of enzyme catalytic reaction and high degradation efficiency, and can not damage the nutrient substances of grains.

To date, there have been some studies on zearalenone degrading enzymes, resulting in enzymes that can degrade zearalenone toxin, which can specifically bind to zearalenone and degrade it. However, few studies have been made on enzymes involved in the degradation of zearalenone in microorganisms obtained by screening.

Disclosure of Invention

The invention aims to provide a zearalenone degrading enzyme and a coding gene thereof, and application of the zearalenone degrading enzyme in hydrolysis of zearalenone and derivatives thereof.

In order to achieve the purpose of the present invention, the inventor does not make extensive efforts through a great number of experimental studies, and finally obtains the following technical scheme:

a zearalenone degrading enzyme, which has an amino acid sequence shown by SEQ ID No.1 in a sequence table; or the degrading enzyme is conservative variant obtained by deletion, substitution, insertion or/and addition of conservative mutation of one to several amino acids on the basis of the amino acid sequence shown in SEQ ID NO. 1.

It should be noted that the zearalenone degrading enzyme provided by the invention is a lactone hydrolase. The amino acid sequence shown in SEQ ID No.1 consists of 266 amino acid residues.

In order to facilitate purification of the above-mentioned degrading enzyme protein, a tag shown in Table 1 may be attached to the amino terminus or the carboxyl terminus of the protein consisting of the above-mentioned amino acid sequence.

TABLE 1 sequences of tags

Label (R)	Residue of	Amino acid sequence
			Poly-Arg	5-6 (typically 5)	RRRRR
Poly-His	2-10 (generally 6)	HHHHHH
			FLAG	8	DYKDDDDK
Strep-tag II	8	WSHPQFEK
			c-myc	10	EQKLISEEDL

The degrading enzyme protein can be artificially synthesized, or can be obtained by synthesizing the coding gene and then carrying out biological expression. The coding gene of the protein can also be obtained by deleting, replacing, inserting or adding one to several amino acid sequences shown in SEQ ID NO.1 and keeping the original enzyme activity, or connecting the coding sequences of the tags shown in the table 1.

A gene encoding zearalenone degrading enzyme, which encodes:

(a) a protein having an amino acid sequence shown in SEQ ID NO. 1; or

(b) A protein having an amino acid sequence shown in SEQ ID No.1 derived from deletion, substitution, insertion or/and addition of one to several amino acids and having a zearalenone and its derivative degrading activity.

It should be noted that the zearalenone and its derivative degrading activity means that it can cleave the lactone bond of the substrate, subsequently produce dihydroxyphenyl derivatives with open side chains and release carbon dioxide to act on several substrates zearalenone, α -zearalenol, β -zearalenol, α -zearalanol, β -zearalanol.

Further, the coding gene of the zearalenone degrading enzyme is a DNA molecule of (i), (ii) or (iii):

(i) a DNA molecule having a nucleotide sequence shown in SEQ ID NO. 2;

(ii) (ii) a DNA molecule which hybridizes under stringent conditions to the nucleotide sequence of (i) and which encodes a protein having the activity of degrading zearalenone and several derivatives thereof;

(iii) (iii) a DNA molecule having a nucleotide sequence having 90% or more homology with the nucleotide sequence described in (i) or (ii).

The nucleotide sequence shown in SEQ ID NO.2 consists of 801 nucleotides.

Further, the stringent condition is a solution with a sodium ion concentration of 50-300mM, and the reaction temperature is 50-68 ℃.

For example: in the molecular hybridization, hybridization may be carried out at 65 ℃ in a solution of 6 XSSC, 0.5% by mass SDS, followed by washing once each of 2 XSSC, 0.1% by mass SDS, 1 XSSC, 0.1% by mass SDS. Wherein the Chinese name of SDS is sodium dodecyl sulfate, and 1 XSSC comprises 0.15mol/L NaCl and 0.015mol/L citric acid; SDS and SSC at various fold concentrations are common reagents in the art.

The recombinant vector, the expression cassette, the transgenic cell line or the recombinant strain containing any one of the coding genes also belong to the protection scope of the invention.

The invention provides a recombinant vector, which comprises the coding gene of the zearalenone degrading enzyme. Specifically, the recombinant vector is a recombinant expression vector obtained by inserting any one of the coding genes into a multiple cloning site of a starting vector (for example, pET28 a). The recombinant expression vector containing the gene can be constructed by using the existing expression vector. When the gene is used for constructing a recombinant expression vector, any enhanced promoter or constitutive promoter can be added before the transcription initiation nucleotide, and the enhanced promoter or constitutive promoter can be used independently or combined with other promoters; in addition, when the gene of the present invention is used to construct a recombinant expression vector, enhancers, including translational or transcriptional enhancers, may be used, and these enhancer regions may be ATG initiation codon or initiation codon of adjacent regions, etc., but must be in the same reading frame as the coding sequence to ensure proper translation of the entire sequence. The translational control signals and initiation codons are widely derived, either naturally or synthetically. The translation initiation region may be derived from a transcription initiation region or a structural gene.

The present invention also provides a transformant comprising the above recombinant vector. The transformant may be a recombinant bacterium obtained by transforming Escherichia coli BL21(DE3) with a recombinant expression vector obtained by inserting any of the above-described encoding genes into a multiple cloning site of a starting vector (e.g., pET28a vector).

The invention also provides a primer pair for amplifying the full length of the coding gene of the zearalenone degrading enzyme and any fragment thereof. For example: the sequences of the primer pairs are shown as SEQ ID NO.3 and SEQ ID NO. 4.

The application of any one of the protein, the coding gene, the recombinant expression vector, the expression cassette, the transgenic cell line or the recombinant bacteria in degrading zearalenone, alpha-zearalenol, beta-zearalenol, alpha-zearalanol and beta-zearalanol also belongs to the protection scope of the invention.

In the course of a particular application, the following method may be employed: taking zearalenone, alpha-zearalenol, beta-zearalenol, alpha-zearalenol and beta-zearalenol as substrates, and carrying out enzymolysis on the zearalenone, the alpha-zearalenol, the beta-zearalenol, the alpha-zearalenol and the beta-zearalenol by using zearalenone degrading enzymes under the condition of alkaline pH.

The enzymolysis conditions comprise: the temperature of the reaction system is 30-40 ℃, preferably 40 ℃, and the pH value of the reaction system is 6.0-9.0, preferably 8.0.

The present invention also provides a method for producing a zearalenone degrading enzyme, which comprises culturing the above transformant and collecting the zearalenone degrading enzyme from the culture product. The collected zearalenone degrading enzyme may be further purified.

Compared with the prior art, the protein provided by the invention has the degradation activity of zearalenone and several derivatives thereof, and belongs to zearalenone degrading enzymes. Compared with other characterized amino acid sequences of zearalenone degrading enzymes, the protein has similarity of no more than 65%, belongs to a brand-new zearalenone degrading enzyme, and provides a new choice for people to degrade zearalenone. The most suitable natural substrate of the zearalenone degrading enzyme is zearalenone, and the zearalenone degrading enzyme has the characteristic of higher activity under the condition of alkaline pH and has better stability under different pH values.

In addition, the zearalenone degrading enzyme Zhd518 provided by the invention has remarkable progress, and is mainly reflected in the following aspects:

(1) the literature reports that the zearalenone degrading enzyme that has been characterized is Zhd101, and the other two are the amino acid sequences of zearalenone degrading enzymes of ZEN-JJM and Zlhy-6, which have 99% and 98% amino acid identity with Zhd101, with essentially identical properties. The amino acid homology of Zhd518 and Zhd101 is only 65 percent, and the enzyme is determined to be a novel zearalenone degrading enzyme. In addition, the optimum reaction temperature of the already characterized zearalenone degrading enzyme Zhd101 was 37 ℃ and the optimum pH was 9.5. The optimum temperature of Zhd518 in the present invention is 40 ℃ and the optimum pH is 8.0, so that it does not require an environment with high alkalinity. Zhd518 has 60% enzyme activity at 30-40 deg.C, and 70% or more enzyme activity at pH 6.0-9.0. This illustrates the significant advantages of Zhd518 resulting from the present invention.

(2) The zearalenone degrading enzyme Zhd518 provided by the invention has degrading activity on ZEN and four derivatives thereof, but has different degrading capabilities. The results are as follows: the diluted enzyme solution is subjected to enzyme activity determination under different substrate conditions with the same concentration (the final substrate concentration in the reaction system is 20.0 mu g/ml). The enzyme activity measured by taking zearalenone as a substrate is taken as a reference (100%), and the relative enzyme activities measured by taking alpha-zearalenol, beta-zearalenol, alpha-zearalanol and beta-zearalanol as substrates are respectively 6.6%, 94.9%, 32.6% and 21.5%. Thus, the enzyme has a higher activity ratio on zearalenone and β -zearalenol, and the other times.

Drawings

FIG. 1 is an SDS-PAGE electrophoresis of zearalenone degrading enzyme Zhd518 protein before and after purification.

FIG. 2 shows the results of the change of the activity of zearalenone degrading enzyme Zhd518 with temperature.

FIG. 3 shows the results of the change of the activity of zearalenone degrading enzyme Zhd518 depending on pH.

FIG. 4 shows the stability of the activity of zearalenone degrading enzyme Zhd518 at different temperatures.

FIG. 5 shows the stability of the activity of zearalenone degrading enzyme Zhd518 at different pH.

Detailed Description

The principles and features of this invention are described below in conjunction with the following drawings, which are set forth by way of illustration only and are not intended to limit the scope of the invention.

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Example 1 preparation and purification of proteins and genes

1. Artificial synthesis of gene sequences

The nucleotide sequence shown in SEQ ID NO.2 was assigned to Wuhan Kingkurui bioengineering, Inc. to perform gene artificial synthesis according to the conventional technique in the art, and the gene was inserted into plasmid vector pUC57 and stored for use.

2. Amplification of Gene sequences

The primer pair is designed according to the nucleotide sequence shown in SEQ ID NO.2 as follows:

a forward primer: 5' -CGCGGATCCATGGCCGCTACACGTACACGAGG-3' as shown in SEQ ID NO. 3;

reverse primer: 5' -CCGCTCGAGCTATTTCAAATACTTCCGACTCG-3' as shown in SEQ ID NO. 4;

the underlined portion of the forward primer is the BamHI site and the underlined portion of the reverse primer is the XhoI site.

And (3) PCR reaction system:

10 × buffer solution	5μL
		dNTP	4μL
ExTaq DNA polymerase	0.5μL
		Forward primer	1μL
Reverse primer	1μL
		Form panel	0.5μL
Water (W)	38μL

And (3) PCR reaction conditions: pre-denaturation at 94 ℃ for 5min, then denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 30s, extension at 72 ℃ for 1min, 30 cycles, and finally extension at 72 ℃ for 10 min.

The PCR product was checked for yield and specificity by agarose gel electrophoresis at 0.7% by mass and purified by a DNA purification kit (ultra-thin centrifugal column type, manufactured by Tiangen Co.). The purified PCR product was sequenced, and as a result, the sequence of the PCR product was found to include the 1-801 positions shown in SEQ ID NO.2 and was named zhd518 DNA fragment.

3. Construction of recombinant expression vectors

1) The PCR product with the correct sequencing is subjected to double digestion by BamHI and XhoI, and the digestion product is recovered by agarose electrophoresis.

2) Plasmid pET28a (Cat. N069864-3, Novogen) was digested simultaneously with BamHI and XhoI, and the digested product was recovered by agarose electrophoresis.

3) The enzyme digestion product of the step 1) and the enzyme digestion product of the step 2) are connected, the connection product is electrically shocked to transform escherichia coli DH5 alpha, then the obtained product is spread on an LB plate containing 50 mu g/mL kanamycin and cultured overnight at 37 ℃, colony PCR is carried out on the obtained transformant by using the forward primer and the reverse primer, recombinant bacteria containing zhd518 gene are screened, a plasmid of the recombinant bacteria is extracted, and sequencing verification is carried out, so that zhd518 DNA fragment is inserted between BamHI enzyme digestion sites and XhoI enzyme digestion sites of pET28a, the fragment comprises nucleotides from 1 to 801 th positions of SEQ ID NO.2 from 5' end, the insertion direction is correct, and the recombinant plasmid is named as pET28a-zhd 518.

4. Preparation of engineering bacteria

The plasmid pET28a-zhd518 was transformed into E.coli BL21(DE3) (Cat. N0CD601, available from Okinawa) by electric shock, and then plated on an LB plate containing 50. mu.g/mL kanamycin, and cultured overnight at 37 ℃ to obtain an engineered bacterium containing the plasmid pET28a-zhd518, which was designated as BL21/pET28a-zhd 518.

Escherichia coli BL21(DE3) was transformed using pET28a in place of pET28a-zhd518, and a recombinant strain containing pET28a was obtained as a control strain in the same manner as above. The positive recombinant strain transformed into BL21(DE3) was designated as BL21/pET28 a.

5. Expression and purification of proteins of interest

His60Ni Superflow resin purification column was purchased from TaKaRa under Cat No. 635660.

GE HiTrap Desainting purification columns were purchased from GE Healthcare under the catalog numbers 17-1408-01 and 17-5053-01, respectively.

Culturing the positive recombinant bacterium BL21/pET28a-zhd518 prepared in the step 4 in an LB culture medium containing 50 mu g/mL kanamycin, and culturing at 37 ℃ for 3 h; OD₆₀₀When 0.7, IPTG was added to a final concentration of 0.8mM in LB medium, and the medium was turned to 18 ℃ for further culture for 16 h.

Centrifuging at 3800rpm for 15min to collect thallus, suspending in PBS (50mM Tris-HCl, pH7.4, 0.5M NaCl), ultrasonic disrupting in ice bath (60w, 10 min; ultrasonic for 1s, stopping for 2s), centrifuging at 12000rpm for 10min to remove cell debris, and collecting supernatant; the supernatant was passed through a His60Ni Superflow resin purification column, washed with 5mL of ultra-pure water, then rinsed with 10mL of solution A (50mM Tris-HCl, pH8.0, 25mM imidazole), and finally eluted with 5mL of solution B (50mM Tris-HCl, pH8.0, 500mM imidazole), and the eluate was collected. The eluate was then desalted by a Desalting column GE HiTrap desaling and eluted with solution C (50mM Tris-HCl, pH 8.0) to give Zhd518 purified enzyme solution.

And (4) culturing and purifying the control bacteria prepared in the step (4) by adopting the same steps, and taking the obtained solution as a control enzyme solution.

SDS-PAGE showed that the molecular weight of the purified Zhd518 protein was approximately 29kDa, corresponding to 29.4kDa as inferred by theory. The results are shown in FIG. 1, lane M shows protein molecular weight standards (250,150, 100,75,50,37,25 kDa); lane 1 shows the supernatant of E.coli BL21/pET28a-zhd518 after disruption; lane 2 shows Zhd518 protein after Ni-NTA column purification; lane 3 shows Zhd518 protein after purification on a GE desaling Desalting column. It can be seen that Zhd518 protein was already obtained. The control group experiment was also performed, but the target protein was not obtained from the control bacteria.

Example 2 verification of protein function Using zearalenone as substrate

The unit of enzyme activity is defined as the amount of enzyme required to degrade 1. mu.g of the substrate zearalenone within 1min as a unit of enzyme activity U.

(one) optimum temperature

The Zhd 518-purified enzyme solution obtained in step 5 of example 1 was diluted with 50mM Tris-HCl buffer solution (pH 8.0), and the enzyme activity was measured using the diluted enzyme solution. The diluted enzyme solution was recorded as a diluted enzyme solution.

The solution A comprises the following components: consists of 50mM Tris-HCl buffer solution with the pH value of 8.0 and zearalenone solution; the final concentration of the substrate zearalenone in 0.5mL of the reaction system was 20.0. mu.g/mL.

Experimental groups: the activity determination reaction system is 0.5mL, and the reaction system consists of 0.45mL of solution A and 0.05mL of diluted enzyme solution; the pH value of the reaction system is 8.0; after the reaction system is incubated for 10min in a specific temperature range (20-55 ℃), 0.5mL of chromatographic grade methanol is used for stopping the reaction, and the degradation amount of the substrate is measured by using a High Performance Liquid Chromatograph (HPLC) after cooling.

The results are shown in FIG. 2. FIG. 2 shows that zearalenone degrading enzymes have activity to degrade zearalenone. The zearalenone degrading enzyme has the highest enzyme activity at 40 ℃; the degradation amount of the substrate zearalenone of the enzyme activity reaction system at the temperature is taken as the relative activity of 100%, and the ratio of the degradation amount of the substrate zearalenone of the enzyme activity reaction system at other temperatures to the degradation amount of the substrate zearalenone of the highest enzyme activity system is taken as the relative activity. Has activity of over 60 percent under the condition of 30-40 ℃.

Control group: the above experiment was carried out using a protein obtained from the control strain BL21/pET28a (referred to as a control enzyme solution), and the control enzyme solution had no activity of degrading zearalenone under any temperature condition.

The experiment was repeated 3 times, and the results were consistent.

(II) optimum pH

The following diluted enzyme solutions were obtained by diluting Zhd518 purified enzyme solution in step 5 of example 1 with the buffer solutions in each group.

Experimental groups: the activity determination reaction system is 0.5mL, and consists of 0.45mL of solution B (B1, B2, B3, B4, B5, B6, B7, B8, B9, B10, B11, B12, B13, B14 and B15) and 0.05mL of diluted enzyme liquid, and the final concentration of the substrate zearalenone in 0.5mL of the reaction system is 20.0 mu g/mL.

Composition of solution B1: 50mM glycine-HCl buffer solution and a substrate zearalenone; the pH of solution B1 was 2.0.

Composition of solution B2: the same composition as in solution B1, except that 50mM Glycine-HCl buffer was replaced with 0.2M Na₂HPO₄-a citric acid buffer. The pH of solution B2 was 3.0.

Composition of solution B3: the same composition as in solution B1, except that 50mM Glycine-HCl buffer was replaced with 0.2M Na₂HPO₄-a citric acid buffer. The pH of solution B3 was 4.0.

Composition of solution B4: the same composition as in solution B1, except that 50mM Glycine-HCl buffer was replaced with 0.2M Na₂HPO₄-a citric acid buffer. The pH of solution B4 was 5.0.

Composition of solution B5: the same composition as in solution B1, except that 50mM Glycine-HCl buffer was replaced with 0.2M Na₂HPO₄-a citric acid buffer. The pH of solution B5 was 6.0.

Composition of solution B6: the same composition as in solution B1, except that 50mM Glycine-HCl buffer was replaced with 0.2M Na₂HPO₄-a citric acid buffer. The pH of solution B6 was 7.0.

Composition of solution B7: the same composition as in solution B1, except that 50mM Glycine-HCl buffer was replaced with 0.2M Na₂HPO₄-a citric acid buffer. The pH of solution B7 was 7.5.

Composition of solution B8: the same composition as in solution B1 was used, except that 50mM Glycine-HCl buffer was replaced with 50mM Tris-HCl buffer. The pH of solution B8 was 7.5.

Composition of solution B9: the same composition as in solution B1 was used, except that 50mM Glycine-HCl buffer was replaced with 50mM Tris-HCl buffer. The pH of solution B9 was 8.0.

Composition of solution B10: the same composition as in solution B1 was used, except that 50mM Glycine-HCl buffer was replaced with 50mM Tris-HCl buffer. The pH of solution B10 was 8.5.

Composition of solution B11: the same composition as in solution B1 was used, except that 50mM Glycine-HCl buffer was replaced with 50mM Tris-HCl buffer. The pH of solution B11 was 9.0.

Composition of solution B12: the same composition as for solution B1, except that 50mM Glycine-HCl buffer was replaced with 50mM Glycine-NaOH buffer. The pH of solution B12 was 9.0.

Composition of solution B13: the same composition as for solution B1, except that 50mM Glycine-HCl buffer was replaced with 50mM Glycine-NaOH buffer. The pH of solution B13 was 9.5.

Composition of solution B14: the same composition as for solution B1, except that 50mM Glycine-HCl buffer was replaced with 50mM Glycine-NaOH buffer. The pH of solution B14 was 10.0.

Composition of solution B15: the same composition as for solution B1, except that 50mM Glycine-HCl buffer was replaced with 50mM Glycine-NaOH buffer. The pH of solution B15 was 11.0.

After incubating the reaction system at 40 ℃ for 10min, 0.5mL of chromatographic grade methanol was added to terminate the reaction, and after cooling, the amount of substrate degradation was determined using High Performance Liquid Chromatography (HPLC).

The experiment was performed in triplicate.

The results are shown in FIG. 3.

The zearalenone degrading enzyme has activity of hydrolyzing zearalenone under the condition that the pH is between 3.0 and 10.0, and can degrade zearalenone.

FIG. 3 shows that zearalenone degrading enzyme has the highest enzyme activity at pH 8.0. The degradation amount of the substrate zearalenone of the highest enzyme activity system is taken as the relative activity of 100%, and the ratio of the degradation amount of the substrate zearalenone of other reaction systems to the degradation amount of the substrate zearalenone of the highest enzyme activity system is taken as the respective relative activity. Has more than 70% activity under the conditions of pH6.0-pH 9.0.

Control group: the above experiment was carried out using a protein obtained from the control bacterium BL21/pET28a (referred to as a control enzyme solution), and the control enzyme solution had no activity of degrading zearalenone under any pH condition.

The experiment was repeated 3 times, and the results were consistent.

(III) thermostability of the enzyme

The Zhd 518-purified enzyme solution obtained in step 5 of example 1 was diluted with 50mM Tris-HCl buffer solution (pH 8.0), and enzyme activity was measured using zearalenone as a substrate. The diluted enzyme solution was recorded as a diluted enzyme solution.

The diluted enzyme solution was allowed to stand in a water bath at 20 ℃ 30 ℃,37 ℃, 40 ℃, 45 ℃ and 50 ℃ for 10 minutes, respectively, and the residual activity of the enzyme was measured. The results show that the enzyme is stable at 20 ℃ to 40 ℃ and loses 80% of its activity by treatment at 45 ℃ for 10 minutes.

(IV) pH tolerance

The diluted enzyme solution is respectively placed for 16 hours under the conditions of pH 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0 and 11.0 and the temperature of 4.0 ℃, and then the zearalenone is used as a substrate to determine the residual enzyme activity. The result shows that more than 60 percent of relative enzyme activity still remains under the condition of pH 6.0-10.0. Indicating that the enzyme has good pH tolerance.

(V) substrate specificity

And (3) respectively carrying out enzyme activity determination on the diluted enzyme solution under different substrate conditions with the same concentration (the final substrate concentration in the reaction system is 20.0 mu g/ml), wherein the substrates are zearalenone, alpha-zearalenol, beta-zearalenol, alpha-zearalanol and beta-zearalanol.

The enzyme activity measured by taking zearalenone as a substrate is taken as a reference (100%), and the relative enzyme activities measured by taking alpha-zearalenol, beta-zearalenol, alpha-zearalanol and beta-zearalanol as substrates are respectively 6.6%, 94.9%, 32.6% and 21.5%. Therefore, the activity of the enzyme on zearalenone and beta-zearalenol is higher.

Example 3 verification of protein function Using beta-zearalenol as substrate

The diluted enzyme solution described below was obtained by diluting Zhd 518-pure enzyme solution in step 5 of example 1 with 50mM Tris-HCl buffer.

Experimental groups: taking beta-zearalenol as a substrate (the final concentration of the substrate in a reaction system is 20.0 mu g/mL), wherein the activity determination reaction system is 0.5mL, and 0.45mL of substrate solution and 0.05mL of diluted enzyme solution are used; the pH value of the reaction system is 8.0; after the reaction system reacts for 10min at the optimum temperature of 40 ℃, 0.5mL of chromatographic grade methanol is used for stopping the reaction, and the degradation amount of the substrate is measured by using a High Performance Liquid Chromatograph (HPLC) after cooling.

The experiment was repeated three times, and the results were consistent.

The result showed that the enzyme activity was 823U/mg when β -zearalenol was used as a substrate at 40 ℃ and pH 8.0.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.

SEQUENCE LISTING

<110> university of Hubei

<120> zearalenone degrading enzyme, and coding gene and application thereof

<160>4

<170>PatentIn version 3.5

<210>1

<211>266

<212>PRT

<213> Artificial sequence

<400>1

Met Ala Ala Thr Arg Thr Arg Gly Tyr Val Thr Thr Lys Asp Gly Ile

1 5 1015

Lys Trp Tyr Tyr Glu Gln Glu Gly Ser Gly Pro Asp Val Val Leu Ile

20 25 30

Pro Asp Gly Leu Gly Glu Cys Gln Met Phe Asp Lys Pro Met Ser Leu

35 40 45

Ile Ala Ser Asn Gly Phe Arg Val Thr Thr Phe Asp Met Pro Gly Met

50 55 60

Ser Arg Ser Ser Asp Ala Pro Pro Glu Thr Tyr Gln Asp Ile Thr Gly

65 70 75 80

Arg Lys Leu Ala Gly Tyr Ile Ile Thr Leu Leu Asp Thr Leu Asp Ile

85 90 95

Lys Ile Ala Ser Val Trp Gly Cys Ser Ser Gly Ala Ser Thr Val Leu

100 105 110

Ala Leu Cys Ser Asp Tyr Pro Glu Arg Val Arg Asn Gly Met Pro His

115 120 125

Glu Val Pro Thr Glu Asn Pro Asp Ile Leu Leu His Ile His Glu Val

130 135 140

Asp Pro Ala Thr Ile Ser Gln Glu Met Ala Ala Asn Ser Arg Ala Tyr

145 150 155 160

Ser Gly Asn Val Glu Ala Trp Asp Ala Leu Gly Pro Glu Val His Ala

165 170 175

Arg Leu His Asp Asn Tyr Pro Arg Trp Ala Tyr Gly Tyr Pro Arg Thr

180 185 190

Ile Pro Pro Ser Ala Pro Val Lys Thr Glu Asp Leu His Lys Val Pro

195 200 205

Ile Asp Trp Thr Val Gly Ala Ser Thr Pro Thr Lys Leu Phe Phe Glu

210 215 220

Asn Ile Val Ile Ala Ala Arg Glu Gly Ile Asn Ile Gly Thr Leu Pro

225 230 235 240

Gly Asn His Phe Pro Tyr Val Ser His Pro Glu Glu Phe Ala Lys Tyr

245 250 255

Val Val Glu Thr Ser Arg Lys Tyr Leu Lys

260 265

<210>2

<211>801

<212>DNA

<213> Artificial sequence

<400>2

atggccgcta cacgtacacg aggatatgtt accactaaag acggaataaa atggtactat 60

gagcaagagg gttccgggcc tgatgttgtc ttgattccag acggcctagg cgaatgtcag 120

atgtttgata agccaatgtc tctaattgcc agcaacggct tcagagtaac gacattcgac 180

atgcctggaa tgtcaagatc atccgatgca ccaccagaaa cgtatcagga cattacaggt 240

cgaaaattag ctggctacat tatcacactg ttggacacac tggacatcaa aatcgcttcg 300

gtctggggct gtagctcagg agcctcgacc gtccttgcac tctgctcaga ctacccagag 360

cgagtccgca atggaatgcc ccacgaggtc ccgacagaaa acccagacat tcttctgcat 420

atccacgagg tcgaccctgc cacaatctcg caagaaatgg cagccaactc gagggcctac 480

tcaggaaatg tagaggcatg ggacgctctc gggccagaag tccacgcaag gcttcatgac 540

aactacccgc gatgggcata tggctatcca cgtaccatac ccccgtccgc gccggtgaaa 600

actgaagatc tacacaaggt tccaatagat tggaccgttg gcgcttcaac tcccaccaag 660

ttgttcttcg aaaacatcgt catcgcggcc agggagggca tcaatattgg aactctgccg 720

ggcaatcatt tcccatatgt ctcgcacccg gaagagtttg cgaagtatgt cgttgagacg 780

agtcggaagt atttgaaata g 801

<210>3

<211>32

<212>DNA

<213> Artificial sequence

<400>3

cgcggatcca tggccgctac acgtacacga gg 32

<210>4

<211>32

<212>DNA

<213> Artificial sequence

<400>4

ccgctcgagc tatttcaaat acttccgact cg 32

Claims

1. The application of the zearalenone degrading enzyme in hydrolyzing zearalenone and derivatives thereof is characterized in that the amino acid sequence of the zearalenone degrading enzyme is shown as SEQ ID NO.1, and the derivatives are alpha-zearalenol, beta-zearalenol, alpha-zearalenol or beta-zearalenol.

2. The use of a zearalenone degrading enzyme according to claim 1 for hydrolyzing zearalenone and its derivatives, wherein the hydrolysis conditions comprise: the temperature of the reaction system is 30-40 ℃, and the pH value of the reaction system is 6.0-9.0.

3. The use of a zearalenone degrading enzyme according to claim 1 for hydrolyzing zearalenone and its derivatives, wherein the hydrolysis conditions comprise: the temperature of the reaction system was 40 ℃ and the pH of the reaction system was 8.0.