CN114045276A

CN114045276A - Neutral zearalenone degrading enzyme mutant with improved specific enzyme activity

Info

Publication number: CN114045276A
Application number: CN202111322894.2A
Authority: CN
Inventors: 张桂敏; 王美星; 李佳维; 李晓迪; 巫攀; 袁其朋
Original assignee: Beijing University of Chemical Technology
Current assignee: Beijing University of Chemical Technology
Priority date: 2021-11-09
Filing date: 2021-11-09
Publication date: 2022-02-15
Anticipated expiration: 2041-11-09
Also published as: CN114045276B

Abstract

The invention belongs to the technical field of enzyme engineering, and discloses a neutral zearalenone degrading enzyme mutant with improved specific enzyme activity. The mutant has an amino acid sequence shown in SEQ ID NO.1, or the mutant 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 specific enzyme activity of the neutral zearalenone degrading enzyme mutant provided by the invention is 3.3 times of that of a wild type, and the mutant can be widely applied to the enzymatic degradation of mycotoxin zearalenone.

Description

Neutral zearalenone degrading enzyme mutant with improved specific enzyme activity

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a neutral zearalenone degrading enzyme mutant with improved specific enzyme activity, a coding gene thereof, and application of the mutant in zearalenone hydrolysis.

Background

Zearalenone (ZEN), also known as F-2 toxin, was originally isolated from corn suffering from head blight and is a nonsteroidal estrogen mycotoxin that can be produced by many fusarium species and is produced by crops both 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.

ZEN is one of the most widespread mycotoxins contaminating food grains in the world, and ZEN is detected in grains and by-products thereof all over the world. ZEN has the characteristics of wide distribution, quick propagation, high toxicity and the like, and can further harm the life health of people and animals by polluting crops. ZEN has a chemical structure similar to natural estrogen and therefore is able to competitively bind to estrogen receptors, causing external and internal genital changes and reproductive disorders, leading to hyperestrogenic symptoms and infertility, and such toxins also stimulate the growth of breast cancer cell lines and are carcinogenic in mice.

In view of the hazards of such toxins, strict standards are set by most countries for ZEN content in food or feed. Due to the high stability of ZEN, the removal of such toxins is extremely inefficient using traditional physical and chemical methods. To address this problem, enzymatic degradation has become a promising strategy to reduce contamination by such toxins. 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 ZEN toxin, which can specifically bind to ZEN and degrade it. The present inventors have studied to obtain three zearalenone degrading enzymes, for example, CN107099520A discloses a zearalenone degrading enzyme Zhd, CN108085306A discloses a zearalenone degrading enzyme zhda Y3 and its mutant, and the present inventors have published papers (Wang M, Yin L, Hu H, Nimal S J, Zhou Y, & Zhang g. expression, functional analysis and mutation of a novel neutral zearalenone-degrading enzyme [ J ]. International Journal of Biological Macromolecules,2018,118: 1284. 1292.), obtained mutant Zhd (N156H), mutant 1M for short, the enzyme activity for zearalenone as a substrate is 1.18 times that of the wild type. However, the specific enzyme activity of the zearalenone degrading enzyme obtained above is still not ideal.

Disclosure of Invention

In view of the defects of the prior art, the invention aims to provide a mutation site which is obviously associated with the improvement of the specific enzyme activity of neutral zearalenone degrading enzyme, a neutral zearalenone degrading enzyme mutant with the improved specific enzyme activity, a coding gene thereof and application thereof in the hydrolysis of zearalenone.

In order to achieve the purpose of the present invention, the inventors have made extensive experimental studies and diligent efforts to finally obtain a neutral zearalenone degrading enzyme mutant with improved specific enzyme activity by using site-directed mutagenesis technology to obtain a gene encoding the mutant based on zearalenone degrading enzyme mutant 1M and then performing recombinant expression on the gene site encoding the mutant. Specifically, the technical scheme of the invention is summarized as follows:

an application of a mutation site in improving the specific activity of a zearalenone degrading enzyme mutant 1M is disclosed, wherein an amino acid sequence of the zearalenone degrading enzyme mutant 1M is shown as SEQ ID NO.6, and the mutation site is positioned at 144 amino acids of the zearalenone degrading enzyme mutant 1M, namely valine at 144 site is mutated into glycine.

In addition, the invention can also carry out site-directed mutagenesis at two positions of N156H and V144G on the basis of the wild zearalenone degrading enzyme, thereby obtaining a neutral zearalenone degrading enzyme mutant with improved specific enzyme activity. Therefore, the invention also protects the application of a mutation site in improving the specific enzyme activity of the wild zearalenone degrading enzyme, wherein the amino acid sequence of the wild zearalenone degrading enzyme is shown as SEQ ID NO.5, the mutation sites are positioned at 144 amino acids and 156 amino acids of the wild zearalenone degrading enzyme, valine at the 144 site is mutated into glycine, and asparagine at the 156 site is mutated into histidine.

A neutral zearalenone degrading enzyme mutant with improved specific enzyme activity, wherein the degrading enzyme has an amino acid sequence shown as 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 neutral zearalenone degrading enzyme mutant provided by the invention is a lactone hydrolase. The amino acid sequence shown in SEQ ID NO.1 consists of 266 amino acid residues, and mainly mutations are carried out on valine VAL at position 144 of a neutral zearalenone degrading enzyme Zhd518 mutant 1M to glycine GLY, and the obtained mutant is Zhd518(N156H/V144G) and is named as 2M.

In order to facilitate purification of the above-mentioned mutant proteins of degrading enzymes, tags shown in Table 1 may be attached to the amino terminus or the carboxyl terminus of the proteins consisting of the above-mentioned amino acid sequences.

TABLE 1 sequences of tags

The degrading enzyme mutant protein can be artificially synthesized, or can be obtained by site-directed mutagenesis method based on wild type neutral zearalenone degrading enzyme Zhd518(CN107099520A) or mutant 1M to obtain a vector containing a coding gene, and then carrying out biological expression. The coding gene of the neutral zearalenone degrading enzyme mutant can also be obtained by deleting, replacing, inserting or adding one or more than one of the amino acid sequences shown in SEQ ID NO.1 and keeping the original enzyme activity, or connecting the coding sequences of the labels shown in the table 1.

In addition, the invention also provides a coding gene of the neutral zearalenone degrading enzyme mutant with improved specific enzyme activity, and the coding gene comprises the following components in percentage by weight:

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

(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 degrading activity.

Further, the coding gene of the zearalenone degrading enzyme mutant 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 encodes a protein having zearalenone degrading activity;

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

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

The invention also provides a recombinant vector which comprises the encoding gene of the zearalenone degrading enzyme mutant. 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, which is used for obtaining the recombinant vector containing the full-length coding gene of the neutral zearalenone degrading enzyme mutant by taking the mutant 1M as a template and performing reverse polymerase chain reaction amplification when the recombinant expression vector is constructed. For example: the sequences of the primer pairs are shown as SEQ ID NO.3 and SEQ ID NO. 4.

The protein, the coding gene, the recombinant expression vector, the expression cassette, the transgenic cell line or the application in degrading zearalenone belong to the protection scope of the invention.

In the course of a particular application, the following method may be employed: the zearalenone is used as a substrate, and the zearalenone is subjected to enzymolysis by using a zearalenone degrading enzyme mutant under the conditions of optimum pH8.0 and optimum temperature of 40 ℃. It should be noted that the enzymatic hydrolysis conditions described in the present invention are the optimum pH and the optimum temperature at pH8.0 and temperature 40 ℃, but all the enzymatic hydrolysis conditions include: the temperature of the reaction system is 30-50 ℃, preferably 40 ℃, and the pH value of the reaction system is 6.0-9.0, preferably 8.0, which belong to the protection scope of the invention.

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

It should be noted that the protein provided by the present invention has zearalenone degrading activity, and is referred to as zearalenone degrading enzyme. The neutral zearalenone degrading enzyme mutant provided by the invention is most suitable for a natural substrate zearalenone, and shows higher specific enzyme activity under the conditions of most suitable pH8.0 and most suitable temperature of 40 ℃.

Compared with the prior art, the neutral zearalenone degrading enzyme mutant provided by the invention has the following advantages and remarkable progress:

in a paper published before the present invention, the inventors obtained a mutant Zhd518(N156H), abbreviated as mutant 1M, which has an enzymatic activity 1.18 times higher for the substrate zearalenone than the wild type. On the basis, the inventor analyzes the 3D structure of the wild neutral zearalenone degrading enzyme Zhd518, finds some sites which can influence the specific enzyme activity, and modifies the neutral zearalenone degrading enzyme gene by a site-directed mutagenesis method to finally obtain the mutant 2M, so that the specific enzyme activity of the encoded neutral zearalenone degrading enzyme mutant 2M is obviously enhanced. Therefore, the neutral zearalenone degrading enzyme mutant 2M provided by the invention is mutated at two fixed points (N156H and V144G) on the basis of zearalenone degrading enzyme Zhd518(CN107099520A), and specific test data show that the specific enzyme activity of the mutant 2M to a substrate zearalenone is 481.8U/mg, which is 3.3 times of that of a wild enzyme. This is a completely new property, which indicates that the mutations involved in the present invention are unique and unique, and have great potential for industrial production applications.

Drawings

FIG. 1 is a SDS-PAGE electrophoresis of purified zearalenone degrading enzyme mutant 2M protein.

FIG. 2 shows the improvement of the enzymatic activity of the zearalenone degrading enzyme mutant 2M relative to the wild type.

FIG. 3 shows the comparison results of the enzyme activities of zearalenone degrading enzyme mutants 1M-V30I, 1M-Y187R, 1M-V207R, 1M-S216Y and 1M-T238Y relative to the wild type.

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. Construction of recombinant expression vectors

The neutral zearalenone degrading enzyme mutant 2M provided by the invention is obtained by site-directed mutagenesis on the basis of a recombinant expression vector pET28a-zhd518(N156H) of a mutant 1M of a neutral zearalenone degrading enzyme Zhd518(CN107099520A), so that the construction of the recombinant expression vector is to perform mutagenesis by reverse polymerase chain reaction amplification of the whole circular plasmid pET28a-zhd518 (N156H). The mutagenesis primers for V144G were: V144G-F (5'ccacgagggcgaccctgccac3') as shown in SEQ ID NO. 3; V144G-R (5'gggtcgccctcgtggatatgcag3') as shown in SEQ ID NO. 4.

PCR system for reverse polymerase chain reaction amplification of genes:

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 7min, 15 cycles, and finally extension at 72 ℃ for 10 min.

The PCR product was checked for yield and specificity by electrophoresis on a 0.7% agarose gel and purified using a DNA purification kit. The purified PCR product was recovered by agarose electrophoresis, and then treated with DpnI enzyme to remove the template strand, E.coli DH 5. alpha. competent cells were transformed by electric shock and plated on LB plate containing 50. mu.g/mL kanamycin, cultured overnight at 37 ℃ and the resulting transformant was subjected to sequencing verification. The result showed that V at position 144 was mutated to G, while the other positions were not mutated, the recombinant vector carried the nucleotide sequence shown in SEQ ID NO.2, the nucleotide sequence shown in SEQ ID NO.2 encoded the mutant shown in SEQ ID NO.1, and the recombinant plasmid was named pET28 a-2M.

It is worth to say that the inventor analyzes the 3D structure of the wild neutral zearalenone degrading enzyme Zhd518 to find out several sites which may affect the specific enzyme activity, and modifies the neutral zearalenone degrading enzyme gene by site-directed mutagenesis method, and the best mutant is 2M. In addition, the experimental sites possibly influencing specific enzyme activity found by the inventor also comprise V30I, Y187R, V207R, S216Y and T238Y, and the sites are respectively obtained by a site-directed mutagenesis method on the basis of the mutant 1M and are respectively named as 1M-V30I, 1M-Y187R, 1M-V207R, 1M-S216Y and 1M-T238Y.

2. Preparation of engineering bacteria

The plasmid pET28a-2M was transformed into E.coli BL21(DE3) (Cat. N0 CD601, all-Kokai Co., Ltd.) by electric shock, and then applied to LB plate containing 50. mu.g/mL kanamycin, and cultured overnight at 37 ℃ to obtain an engineered bacterium containing the plasmid pET28a-2M, which was designated as BL21/pET28 a-2M.

Escherichia coli BL21(DE3) was transformed with pET28a in place of pET28a-2M, and the procedure was as above, to obtain a recombinant bacterium containing pET28a as a control bacterium. The positive recombinant strain transformed into BL21(DE3) was designated as BL21/pET28 a.

3. Expression and purification of proteins of interest

His60 Ni Superflow resin purification column was purchased from TaKaRa under Cat No. 635660.

GE HiTrap Desainting purification columns were purchased from GE Healthcare under catalog number 17-1408-01, respectively.

Culturing the positive recombinant bacterium BL21/pET28a-2M prepared in the step 2 in an LB culture medium containing 50 mu g/mL kanamycin, and culturing at 37 ℃ for 3 h; OD₆₀₀When equal to 0.7IPTG was added to a final concentration of 0.8mM in LB medium, and the medium was turned to 18 ℃ to continue the culture for 16 hours.

Centrifuging at 3800rpm for 15min, collecting thallus, suspending in buffer solution A (50mM Tris-HCl buffer, pH8.0), 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 His60 Ni Superflow resi n purification column, washed with 5mL of ultra-pure water, then washed with 10mL of solution B (50mM Tris-HCl buffer, pH8.0, 25mM imidazole, 150mM NaCl), and finally eluted with 5mL of solution C (50mM Tris-HCl buffer, pH8.0, 500mM imidazole, 150mM NaCl), and the eluate was collected. Then, the eluate was desalted by a Desalting column GE HiTrap desaling and eluted with solution A (50mM Tris-HCl buffer, pH8.0) to give 2M pure enzyme solution.

And (3) culturing and purifying the control bacteria prepared in the step (2) by adopting the same steps, and taking the obtained solution as a control enzyme solution.

SDS-PAGE showed the molecular weight of the purified 2M protein to be about 30kDa, corresponding to 29.3kDa 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, 15,10 kDa); lane 1 shows 2M protein obtained by purifying the supernatant of Escherichia coli BL21/pET28a-2M after disruption with Ni-NTA column and GE desaling Desalting column, and shows that 2M protein was obtained. The control group experiment was also performed, but the target protein was not obtained from the control bacteria.

It is worth mentioning that the mutants obtained from the experimental sites (V30I, Y187R, V207R, S216Y, T238Y) are 1M-V30I, 1M-Y187R, 1M-V207R, 1M-S216Y, 1M-T238Y, respectively. The same recombinant expression vector construction, the preparation of engineering bacteria and the expression and purification of target protein are carried out.

Example 2 comparison of the specific enzyme Activity of the neutral zearalenone degrading enzyme mutant 2M with that of the wild-type neutral zearalenone degrading enzyme protein

Method for determining activity of zearalenone degrading enzyme

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.

The 2M pure enzyme solution in step 5 of example 1 was diluted with 50mM Tris-HCl buffer solution, pH8.0, 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 50mM 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 was incubated at 40 ℃ for 10min, 0.5mL of chromatographic grade methanol was used to stop the reaction, and the amount of substrate degradation was measured by High Performance Liquid Chromatography (HPLC) after cooling.

Second, protein concentration determination method

Pure BSA calf serum Protein was taken and prepared as a 0.5mg/mL Protein solution according to the BIO-RAD Quick Start TM Bradford Protein Assay Kit (available from BIO-RAD, Inc., cat # 5000201) using instructions, according to its purity, in 50mM Tris-HCl buffer at pH 8.0. Sucking

standard protein solution

0,1, 2, 4, 8, 12, 16 and 20 μ L, and diluting to 20 μ L with 50mM Tris-HCl buffer solution with pH 8.0; the sample protein was mixed with 50mM Tris-HCl buffer, pH8.0, in a ratio to give a total volume of 20. mu.L. 5 mu L of protein solution is taken to react with 200 mu L of 1x dye reagent for 5min, the absorbance OD value is measured at 595nm, and a standard curve of the protein concentration and the OD595 is drawn.

Third, comparison of specific enzyme activities

The specific enzyme activity is obtained by dividing the enzyme activity measured by experiments by the protein concentration, and the experimental result is shown in figure 2, and figure 2 shows that the zearalenone degrading enzyme mutant 2M has the activity of degrading zearalenone. Under the conditions of pH8.0 and 40 ℃, the degradation amount of wild zearalenone degrading enzyme protein Zhd518 in a substrate of an enzyme reaction system is taken as 100 percent of relative activity, and the ratio of the degradation amount of the substrate zearalenone of the mutant 2M in the enzyme activity reaction system to the degradation amount of the substrate zearalenone of the wild protein in the enzyme activity reaction system is taken as the relative activity. The results show that: the enzymatic activity of zearalenone degrading enzyme mutant 2M was 3.3 times that of the wild type at pH8.0 and 40 ℃ as compared with that of the wild type (FIG. 2).

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 result showed that the control enzyme solution had no zearalenone degrading activity.

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

The zearalenone degrading enzyme mutants obtained at experimental sites (V30I, Y187R, V207R, S216Y and T238Y) are 1M-V30I, 1M-Y187R, 1M-V207R, 1M-S216Y and 1M-T238Y respectively. The results of the enzyme activity comparison of the sites show that compared with wild type or mutant 1M, the specific enzyme activity is not obviously improved, even the enzyme activity is reduced. The specific results show that: the specific enzyme activities of zearalenone degrading enzyme mutants 1M-V30I, 1M-Y187R, 1M-V207R, 1M-S216Y, and 1M-T238Y on the substrate zearalenone were 90%, 63%, 66%, 89%, and 64% of the wild type at pH8.0 and 40 deg.C (FIG. 3).

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

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Claims

1. An application of a mutation site in improving the specific activity of a zearalenone degrading enzyme mutant 1M is disclosed, wherein an amino acid sequence of the zearalenone degrading enzyme mutant 1M is shown as SEQ ID NO.6, and the mutation site is positioned at 144 amino acids of the zearalenone degrading enzyme mutant 1M, namely valine at 144 site is mutated into glycine.

2. The application of the mutation site in improving the specific enzyme activity of the wild zearalenone degrading enzyme is characterized in that the amino acid sequence of the wild zearalenone degrading enzyme is shown as SEQ ID No.5, the mutation site is located at 144 amino acids and 156 amino acids of the wild zearalenone degrading enzyme, valine at the 144 site is mutated into glycine, and asparagine at the 156 site is mutated into histidine.

3. A neutral zearalenone degrading enzyme mutant with improved specific enzyme activity is characterized in that the degrading enzyme mutant has an amino acid sequence shown in SEQ ID No. 1; or the degrading enzyme mutant 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.

4. A coding gene of neutral zearalenone degrading enzyme mutant with improved specific enzyme activity is characterized in that the coding gene is as follows: (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 an activity of degrading zearalenone.

5. The gene encoding a neutral zearalenone degrading enzyme mutant having improved specific enzyme activity according to claim 4, wherein the gene 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 zearalenone degrading activity;

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

6. The gene encoding a neutral zearalenone degrading enzyme mutant having improved specific enzyme activity according to claim 5, wherein the stringent conditions are: the reaction temperature is 50-68 ℃ in a solution with the sodium concentration of 50-300 mM.

7. A recombinant vector comprising the gene encoding the zearalenone degrading enzyme mutant of any one of claims 4 to 6.

8. A primer pair, which is used for obtaining the recombinant vector of claim 7 by reverse polymerase chain reaction amplification during the construction of the recombinant expression vector, and the sequences of the primer pair are shown as SEQ ID NO.3 and SEQ ID NO. 4.

9. Use of the neutral zearalenone degrading enzyme mutant according to claim 3 for enzymolysis of zearalenone and improvement of specific enzyme activity.

10. Use according to claim 9, characterized in that the enzymatic conditions comprise: the temperature of the reaction system is 30-50 ℃, and the pH value of the reaction system is 6.0-9.0.