CN107099520A - A kind of zearalenone digestive enzyme and its encoding gene and application - Google Patents

Abstract

The present invention relates to a kind of zearalenone digestive enzyme and its encoding gene, the digestive enzyme has the amino acid sequence shown in SEQ ID NO.1, or the digestive enzyme is the conservative variant for being lacked on the basis of the amino acid sequence shown in SEQ ID NO.1, replacing, insert or/and adding a conservative mutation to several amino acid and obtain.Zearalenone digestive enzyme of the present invention has the advantages that high enzyme activity, pH better tolerances, the enzymolysis of zearalenone and its several derivatives is can be widely applied to, it is relatively good particularly with the substrate specificity of zearalenone and β zearalenols.

Description

A kind of zearalenone degrading enzyme and its coding gene and application

technical field

The invention belongs to the field of biotechnology, and in particular relates to a zearalenone degrading enzyme, its coding gene and its application.

Background technique

Zearalenone, first isolated from corn, is a non-steroidal estrogenic mycotoxin that can be produced by many Fusarium species, both before and after harvest. Zearalenone is consistently found in many crop and cereal by-products including corn, barley, wheat, etc., especially in environments that are suitable for fungal growth.

There are many derivatives of zearalenone, such as zearalenol, which will enter the food chain through contaminated crops and accumulate in human and animal bodies, causing damage to organisms. The chemical structure of zearalenone and its derivatives is similar to natural estrogen, so they can competitively bind to estrogen receptors, causing external and internal genital changes and reproductive disorders, leading to hyperoestrogenism and infertility, The toxins also stimulated the growth of breast cancer cell lines and caused cancer in mice.

In view of the hazards of such toxins, the content of zearalenone, etc. in grains, food and feed must be lower than a certain standard. Since zearalenone etc. is extremely stable, it is inefficient to remove such toxins using traditional physical and chemical methods. To address these issues, a promising strategy to reduce such toxin contamination is enzymatic degradation. Enzyme degradation can not only efficiently convert toxins into non-toxic products, which is safe and environmentally friendly, but also has strong specificity of enzyme-catalyzed reactions, high degradation efficiency, and will not destroy the nutrients of grains.

So far, there have been some studies on zearalenone-degrading enzymes, and some enzymes that can degrade zearalenone toxin have been obtained, and they can specifically bind zearalenone and degrade it. However, there are not many studies on the enzymes related to the degradation of zearalenone in the screened microorganisms.

Contents of the invention

The object of the present invention is to provide a zearalenone degrading enzyme and its coding gene, and its application in hydrolyzing zearalenone and its derivatives.

In order to achieve the purpose of the present invention, the inventor has made unremitting efforts through a large number of experimental studies, and finally obtained the following technical solutions:

A zearalenone degrading enzyme, the degrading enzyme has the amino acid sequence shown in SEQ ID NO.1 in the sequence listing; or the degrading enzyme is based on the amino acid sequence shown in SEQ ID NO.1 deletion, replacement, A conservative variant obtained by inserting or/and adding one to several amino acid conservative mutations.

It should be noted that the zearalenone degrading enzyme provided by the present 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 the purification of the above-mentioned degrading enzyme protein, the tags shown in Table 1 can be attached to the amino-terminus or carboxy-terminus of the protein composed of the above-mentioned amino acid sequence.

Table 1 Sequence of tags

Label Residues amino acid sequence Poly-Arg 5-6 (usually 5) RRRRR Poly-His 2-10 (usually 6) HHHHHH FLAG 8 DYKDDDDK Strep-tag II 8 WSHPQFEK c-myc 10 EQKLISEEDL

The above-mentioned degrading enzyme protein can be synthesized artificially, or its coding gene can be firstly synthesized and then biologically expressed. The protein-encoding gene mentioned above can also be deleted, substituted, inserted or added one to several in the amino acid sequence shown in SEQ ID NO. The coding sequence was obtained.

A gene encoding zearalenone degrading enzyme, the gene encodes:

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

(b) A protein having the 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 zearalenone and its derivatives degrading activity.

It should also be noted that the degradative activity of zearalenone and its derivatives refers to the ability to cleave the lactone bond of the substrate, followed by the generation of dihydroxyphenyl derivatives with open side chains and the release of carbon dioxide, which acts on Gibberella zearalenone enone, α-zearalenol, β-zearalenol, α-zearalenol, β-zearalenol these several substrates.

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

(i) DNA molecule with the nucleotide sequence shown in SEQ ID NO.2;

(ii) a DNA molecule that hybridizes to the nucleotide sequence described in (i) under stringent conditions and encodes a protein with degradative activity of zearalenone and several derivatives thereof;

(iii) A DNA molecule having a nucleotide sequence that is 90% or more homologous to the nucleotide sequence described in (i) or (ii).

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

Further, the stringent conditions are that the reaction temperature is 50-68° C. in a solution with a sodium ion concentration of 50-300 mM.

For example: in the process of molecular hybridization, it can be hybridized at 65°C in a solution of 6×SSC and 0.5% SDS by mass fraction, and then use 2×SSC, 0.1% SDS by mass fraction and 1× The membrane was washed once with SSC and 0.1% SDS respectively. The Chinese name of SDS is sodium dodecyl sulfate, and 1×SSC includes 0.15mol/L NaCl and 0.015mol/L citric acid; SDS and SSC with different concentration multiples are commonly used reagents in this field.

Recombinant vectors, expression cassettes, transgenic cell lines or recombinant bacteria containing any of the above-mentioned coding genes also belong to the protection scope of the present invention.

The present invention provides a recombinant vector, which comprises the coding gene of the above-mentioned zearalenone degrading enzyme. Specifically, the recombinant vector is a recombinant expression vector obtained by inserting any of the above-mentioned coding genes into the multiple cloning site of the starting vector (for example: pET28a). An existing expression vector can be used to construct a recombinant expression vector containing the gene. When using the gene to construct a recombinant expression vector, any enhanced promoter or constitutive promoter can be added before its transcription initiation nucleotide, and they can be used alone or in combination with other promoters; in addition, When using the gene of the present invention to construct a recombinant expression vector, enhancers can also be used, including translation enhancers or transcription enhancers, and these enhancer regions can be ATG start codons or adjacent region start codons, etc., but must be consistent with the coding The reading frames of the sequences are identical to ensure correct translation of the entire sequence. The sources of the translation control signals and initiation codons are extensive and can be natural or synthetic. The translation initiation region can be from a transcription initiation region or a structural gene.

The present invention also provides a transformant comprising the above-mentioned recombinant vector. The transformant can be a recombinant bacterium. For example, the recombinant expression vector obtained by inserting any of the above-mentioned coding genes into the multiple cloning site of the starting vector (for example: pET28a vector) is transformed into Escherichia coli BL21 (DE3) to obtain a recombinant bacterium.

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

Any of the above-mentioned proteins, any of the above-mentioned coding genes, any of the above-mentioned recombinant expression vectors, the expression cassettes, transgenic cell lines or recombinant bacteria can degrade zearalenone, α- The application of several substrates of zearalenol, β-zearalenol, α-zearalenol and β-zearalenol also belongs to the protection scope of the present invention.

In the process of specific application, the following method can be adopted: with zearalenone, α-zearalenol, β-zearalenol, α-zearalenol, β-zearalenol Several kinds of substrates, under alkaline pH conditions, use zearalenone degrading enzymes to zearalenone, α-zearalenol, β-zearalenol, α-zearalenol , β-zearalanol for enzymatic hydrolysis.

The enzymolysis conditions include: the temperature of the reaction system is 30-40°C, preferably 40°C, and the pH of the reaction system is 6.0-9.0, preferably 8.0.

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

Compared with the prior art, the protein provided by the invention has the degrading activity of zearalenone and several derivatives thereof, and belongs to zearalenone degrading enzymes. And compared with the amino acid sequence of other characterized zearalenone degrading enzymes, the similarity of the protein is not more than 65%. a new choice. The most suitable natural substrate of the zearalenone degrading enzyme of the present invention is zearalenone, and has the characteristic of higher activity under partial alkaline pH conditions, and at the same time, has better stability under different pH conditions.

In addition, the zearalenone-degrading enzyme Zhd518 provided by the present invention has significant progress, which is also mainly reflected in the following aspects:

(1) According to literature reports, the zearalenone-degrading enzyme that has been characterized is Zhd101, and the amino acid sequences of the other two zearalenone-degrading enzymes ZEN-JJM and Zlhy-6 are identical to those of Zhd101 99% and 98%, the properties are basically the same. The amino acid homology between Zhd518 and Zhd101 in the present invention is only 65%, and it is determined to be a new type of zearalenone degrading enzyme. In addition, the optimum reaction temperature of the characterized zearalenone-degrading enzyme Zhd101 is 37°C and the optimum pH is 9.5. The optimal temperature of Zhd518 in the present invention is 40°C and the optimal pH is 8.0, so that it does not require a highly alkaline environment. Zhd518 still has 60% enzyme activity in the temperature range of 30-40°C, and has more than 70% enzyme activity in the pH range of 6.0-9.0. This shows that the Zhd518 obtained in the present invention has significant advantages.

(2) The zearalenone-degrading enzyme Zhd518 provided by the present invention has degrading activity on ZEN and four derivatives thereof, but the degrading ability is different. The results are as follows: the enzyme activity was measured under different substrate conditions with the same concentration (the final substrate concentration in the reaction system was 20.0 μg/ml) of the diluted enzyme solution. The enzyme activity measured with zearalenone as the substrate is the reference (100%), with α-zearalenol, β-zearalenol, α-zearalenol, β-zearalenol Alcohol as the substrate measured relative enzyme activities were 6.6%, 94.9%, 32.6%, 21.5%. Therefore, the activity of the enzyme on zearalenone and β-zearalenol is relatively high, and the others are next.

Description of drawings

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

Figure 2 is the result of the change of the activity of zearalenone degrading enzyme Zhd518 with temperature.

Fig. 3 is the result of the change of the activity of zearalenone degrading enzyme Zhd518 with pH.

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

Figure 5 shows the stability of the activity of zearalenone degrading enzyme Zhd518 at different pHs.

detailed description

The principles and features of the present invention are described below in conjunction with the accompanying drawings, and the examples given are only used to explain the present invention, and are not intended to limit the scope of the present invention.

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

The materials and reagents used in the following examples can be obtained from commercial sources unless otherwise specified.

Embodiment 1, preparation and purification of protein and gene

1. Artificial synthesis of gene sequence

The nucleotide sequence shown in SEQ ID NO.2 was entrusted to Wuhan Jinkairui Bioengineering Co., Ltd. to artificially synthesize the gene according to the conventional technology in the field, and the gene was inserted into the plasmid vector pUC57, and stored for future use.

2. Gene sequence amplification

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

Forward primer: 5′-CGC GGATCC ATGGCCGCTACACGTACACGAGG-3′, as shown in SEQ ID NO.3;

Reverse primer: 5'-CCG CTCGAG CTATTTCAAATACTTCCGACTCG-3', as shown in SEQ ID NO.4;

The underlined part of the forward primer is the restriction site of BamHI, and the underlined part of the reverse primer is the restriction site of XhoI.

PCR reaction system:

10× buffer 5μL dNTP 4μL exTaq DNA Polymerase 0.5μL forward primer 1μL reverse primer 1μL template 0.5μL water 38μL

PCR reaction conditions: pre-denaturation at 94°C for 5 min, then denaturation at 94°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 1 min, 30 cycles, and finally extension at 72°C for 10 min.

The yield and specificity of the PCR product were detected by agarose gel electrophoresis with a mass fraction of 0.7%, and purified with a DNA purification kit (ultra-thin spin column type, produced by Tiangen Company). The purified PCR product was sequenced, and the result showed that the sequence of the PCR product included positions 1-801 shown in SEQ ID NO.2, and it was named zhd518 DNA fragment.

3. Construction of recombinant expression vector

1) The above-mentioned PCR products with correct sequencing were digested with BamHI and XhoI, and the digested products were recovered by agarose electrophoresis.

2) Plasmid pET28a (Cat.N0 69864-3, Novogen) was double digested with BamHI and XhoI, and the digested product was recovered by agarose electrophoresis.

3) Ligate the digested product of step 1) and the digested product of step 2), transform the ligated product into Escherichia coli DH5α by electroporation, spread it on an LB plate containing 50 μg/mL kanamycin, and culture it overnight at 37°C. The obtained transformants were subjected to colony PCR with the above-mentioned forward primer and reverse primer, and the recombinant bacteria containing the zhd518 gene were screened, the plasmid of the recombinant bacteria was extracted, and the sequencing verification was carried out. The results showed that at the BamHI and XhoI restriction sites of pET28a The zhd518 DNA fragment was inserted between them, and the fragment included the 1st to 801st nucleotides from the 5' end of SEQ ID NO.2, and the insertion direction was correct. The recombinant plasmid was named pET28a-zhd518.

4. Preparation of engineering bacteria

The plasmid pET28a-zhd518 was transformed into Escherichia coli BL21(DE3) (Cat.NOCD601, Quanshijin Company) by electroporation, and spread on an LB plate containing 50 μg/mL kanamycin, and cultured overnight at 37°C to obtain the plasmid containing pET28a-zhd518 The engineered bacterium, denoted as BL21/pET28a-zhd518.

Replace pET28a-zhd518 with pET28a, transform Escherichia coli BL21(DE3), and follow the same steps as above to obtain a recombinant bacterium containing pET28a as a control bacterium. The positive recombinant bacteria transformed into BL21(DE3) were recorded as BL21/pET28a.

5. Expression and purification of target protein

His60Ni Superflow resin purification column was purchased from TaKaRa Company, the product catalog number is 635660.

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

Culture the positive recombinant bacteria BL21/pET28a-zhd518 prepared in the above step 4 in LB medium containing 50 μg/mL kanamycin, culture at 37°C for 3 hours; when OD ₆₀₀ =0.7, add IPTG to its LB medium The final concentration was 0.8mM, and transferred to 18°C to continue culturing for 16h.

The cells were collected by centrifugation at 3800rpm for 15min, suspended in PBS solution (50mM Tris-HCl, pH7.4, 0.5M NaCl), ultrasonically disrupted in an ice bath (60w, 10min; ultrasonication for 1s, stop for 2s), and then Centrifuge at 12000rpm for 10min to remove cell debris, take the supernatant; pass the supernatant through a His60Ni Superflow resin purification column, rinse with 5mL ultrapure water, and then rinse with 10mL solution A (50mM Tris-HCl, pH8.0, 25mM imidazole), Finally, it was eluted with 5 mL solution B (50 mM Tris-HCl, pH 8.0, 500 mM imidazole), and the eluate was collected. Then the eluate was desalted with desalting column GE HiTrap Desalting, and eluted with solution C (50mM Tris-HCl, pH 8.0) to obtain Zhd518 pure enzyme solution.

The control bacteria prepared in step 4 were cultivated and purified in the same steps, and the obtained solution was used as the control enzyme solution.

SDS-PAGE electrophoresis showed that the molecular weight of the purified Zhd518 protein was about 29kDa, which was in line with the theoretical deduction of 29.4kDa. The results are shown in Figure 1. In Figure 1, swimming lane M represents the protein molecular weight standard (250,150,100,75,50,37,25kDa); 2 represents Zhd518 protein purified by Ni-NTA column; lane 3 represents Zhd518 protein purified by GE Desalting column. It can be seen that the Zhd518 protein has been obtained. At the same time, the experiment of the control group was carried out, but the control bacteria did not obtain the target protein.

Example 2. Verification of protein function using zearalenone as a substrate

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

(1) Optimum temperature

The Zhd518 pure enzyme solution in Step 5 of Example 1 was diluted with 50 mM Tris-HCl buffer solution of pH 8.0, and the enzyme activity was measured with the diluted enzyme solution. The diluted enzyme solution was recorded as the diluted enzyme solution.

The composition of solution A: consists of 50 mM, pH 8.0 Tris-HCl buffer solution and zearalenone solution; the final concentration of the substrate zearalenone in the reaction system 0.5 mL is 20.0 μg/ml.

Experimental group: The reaction system for activity determination is 0.5mL, diluted with 0.45mL solution A and 0.05mL enzyme solution; the pH value of the reaction system is 8.0; mL of chromatographic grade methanol was used to terminate the reaction, and after cooling, the amount of degradation of the substrate was determined by high performance liquid chromatography (HPLC).

The result is shown in Figure 2. Figure 2 shows that the zearalenone degrading enzyme has the activity of degrading zearalenone. At 40°C, the zearalenone-degrading enzyme has the highest enzyme activity; the substrate zearalenone degradation amount of the enzyme activity reaction system at this temperature is regarded as 100% of the relative activity, and the enzyme activity reaction at other temperatures The ratio of the degradation amount of the substrate zearalenone in the system to the degradation amount of the substrate zearalenone in the system with the highest enzyme activity was used as the relative activity. All have an activity of more than 60% under the condition of 30-40°C.

Control group: the above experiment was carried out with the protein obtained from the control bacteria BL21/pET28a (referred to as the control enzyme solution). As a result, the control enzyme solution had no activity of degrading zearalenone no matter under which temperature conditions.

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

(2) Optimum pH

The diluted enzyme solutions in the following groups were obtained by diluting the Zhd518 pure enzyme solution in Step 5 of Example 1 with the buffer solution in each group.

Experimental group: The reaction system for activity determination was 0.5mL, which was composed of 0.45mL solution B (B1, B2, B3, B4, B5, B6, B7, B8, B9, B10, B11, B12, B13, B14 and B15) and 0.05 mL diluted enzyme solution, the final concentration of the substrate zearalenone in the reaction system 0.5mL is 20.0μg/ml.

Composition of solution B1: 50 mM glycine-HCl buffer solution and substrate zearalenone; pH value of solution B1 is 2.0.

Composition of solution B2: the same composition as solution B1, except that 50 mM Glycine-HCl buffer was replaced by 0.2M Na ₂ HPO ₄ -citric acid buffer. Solution B2 has a pH of 3.0.

Composition of solution B3: the same composition as solution B1, except that 50 mM Glycine-HCl buffer was replaced by 0.2M Na ₂ HPO ₄ -citric acid buffer. Solution B3 had a pH of 4.0.

Composition of solution B4: the same composition as solution B1, except that 50 mM Glycine-HCl buffer was replaced by 0.2M Na ₂ HPO ₄ -citric acid buffer. Solution B4 had a pH of 5.0.

Composition of solution B5: the same composition as solution B1, except that 50 mM Glycine-HCl buffer was replaced by 0.2M Na ₂ HPO ₄ -citric acid buffer. Solution B5 has a pH of 6.0.

Composition of solution B6: the same composition as solution B1, except that 50 mM Glycine-HCl buffer was replaced by 0.2M Na ₂ HPO ₄ -citric acid buffer. Solution B6 had a pH of 7.0.

Composition of solution B7: the same composition as solution B1, except that 50 mM Glycine-HCl buffer was replaced by 0.2M Na ₂ HPO ₄ -citric acid buffer. Solution B7 has a pH of 7.5.

Composition of solution B8: the same composition as solution B1, except that 50 mM Glycine-HCl buffer was replaced by 50 mM Tris-HCl buffer. Solution B8 has a pH of 7.5.

Composition of solution B9: the same composition as solution B1, except that 50 mM Glycine-HCl buffer was replaced by 50 mM Tris-HCl buffer. Solution B9 had a pH of 8.0.

Composition of solution B10: the same composition as solution B1, except that 50 mM Glycine-HCl buffer was replaced by 50 mM Tris-HCl buffer. Solution B10 has a pH of 8.5.

Composition of solution B11: the same composition as solution B1, except that 50 mM Glycine-HCl buffer was replaced by 50 mM Tris-HCl buffer. Solution B11 has a pH of 9.0.

Composition of solution B12: the same composition as solution B1, except that 50 mM Glycine-HCl buffer was replaced by 50 mM glycine-NaOH buffer. Solution B12 had a pH of 9.0.

Composition of solution B13: the same composition as solution B1, except that 50 mM Glycine-HCl buffer was replaced by 50 mM glycine-NaOH buffer. Solution B13 had a pH of 9.5.

Composition of solution B14: the same composition as solution B1, except that 50 mM Glycine-HCl buffer was replaced by 50 mM glycine-NaOH buffer. Solution B14 had a pH of 10.0.

Composition of solution B15: the same composition as solution B1, except that 50 mM Glycine-HCl buffer was replaced by 50 mM glycine-NaOH buffer. Solution B15 had a pH of 11.0.

After incubating the reaction system at 40° C. for 10 min, 0.5 mL of chromatographic grade methanol was added to terminate the reaction, and after cooling, the amount of degradation of the substrate was measured by high performance liquid chromatography (HPLC).

The experiment was repeated three times.

The result is shown in Figure 3.

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

Figure 3 shows that the zearalenone degrading enzyme has the highest enzyme activity at pH 8.0. The substrate zearalenone degradation amount of this highest enzyme activity system is regarded as relative activity 100%, the substrate zearalenone degradation amount of other reaction systems is the same as the substrate zearalenone degradation amount of this highest enzyme activity system The ratio of the amount was taken as the relative activity of each. Under the conditions of pH 6.0-pH 9.0, they all have more than 70% activity.

Control group: the above experiment was carried out with the protein obtained from the control bacteria BL21/pET28a (referred to as the control enzyme solution). As a result, the control enzyme solution had no activity of degrading zearalenone no matter under which pH conditions.

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

(3) Enzyme thermostability

The Zhd518 pure enzyme solution in Step 5 of Example 1 was diluted with 50 mM Tris-HCl buffer solution of pH 8.0, and the enzyme activity was measured with the diluted enzyme solution using zearalenone as a substrate. The diluted enzyme solution was recorded as the diluted enzyme solution.

Place the diluted enzyme solution in water baths at 20°C, 30°C, 37°C, 40°C, 45°C, and 50°C for 10 minutes respectively to measure the residual activity of the enzyme. The results showed that the enzyme was stable at 20°C to 40°C, and lost 80% of its activity at 45°C for 10 minutes.

(4) pH tolerance

Place the diluted enzyme solution at pH 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, and 11.0, and place it at 4.0°C for 16 hours, then use zearalenone as a substrate to determine the residual enzyme live. The results showed that more than 60% of the relative enzyme activity still remained under the condition of pH 6.0-10.0. It shows that the enzyme has good pH tolerance.

(5) Substrate specificity

The diluted enzyme solution was tested for enzyme activity under different substrate conditions at the same concentration (the final substrate concentration in the reaction system was 20.0 μg/ml), and the substrates were zearalenone and α-zearalenol respectively. , β-zearalenol, α-zearalenol, β-zearalenol.

The enzyme activity measured with zearalenone as the substrate is the reference (100%), with α-zearalenol, β-zearalenol, α-zearalenol, β-zearalenol Alcohol as the substrate measured relative enzyme activities were 6.6%, 94.9%, 32.6%, 21.5%. Therefore, the enzyme has relatively high activity on zearalenone and β-zearalenol.

Example 3. Verification of protein function using β-zearalenol as a substrate

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

The diluted enzyme solution described below is obtained by diluting the Zhd518 pure enzyme solution in Step 5 of Example 1 with 50 mM Tris-HCl buffer solution.

Experimental group: β-zearalenol was used as the substrate (the final concentration of the substrate in the reaction system was 20.0 μg/ml), the activity assay reaction system was 0.5mL, and 0.45mL substrate solution and 0.05mL diluted enzyme solution; the pH value of the reaction system was 8.0; after the reaction system was reacted at an optimum temperature of 40° C. for 10 min, 0.5 mL of chromatographic grade methanol was used to terminate the reaction, and after cooling, the amount of degradation of the substrate was measured using high performance liquid chromatography (HPLC).

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

The results showed that under the conditions of 40°C and pH 8.0, the enzyme activity measured with β-zearalenol as the substrate was 823U/mg.

The above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included in the protection of the present invention. within range.

SEQUENCE LISTING

<110> Hubei University

<120> A Zearalenone Degrading Enzyme, Its Encoding Gene and Application

<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 10 15

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 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 aggggagggca 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 (10)

1. a kind of zearalenone digestive enzyme, it is characterised in that the digestive enzyme has the amino acid sequence shown in SEQ ID NO.1 Row；Or the digestive enzyme is to lack, replace on the basis of the amino acid sequence shown in SEQ ID NO.1, inserting or/and addition one To several amino acid conservative mutation and the conservative variant that obtains.

2. a kind of encoding gene of zearalenone digestive enzyme, it is characterised in that the gene code：(a) there is SEQ ID The protein of amino acid sequence shown in NO.1；Or (b) have derived from missing, replace, insertion or/and addition one be to several Amino acid sequence shown in the SEQ ID NO.1 of amino acid and the active egg with degrading zearalenone and its derivative White matter.

3. a kind of encoding gene of zearalenone digestive enzyme according to claim 2, it is characterised in that the gene is (i), (ii) or (iii) DNA molecular：

(i) there is the DNA molecular of the nucleotide sequence shown in SEQ ID NO.2；

(ii) under strict conditions with the nucleotide sequence hybridization described in (i) and coding with degrading zearalenone and its spreading out Biological active protein DNA molecule；

(iii) there is the nucleotide sequence of more than 90% homology with the nucleotide sequence of (i) or (ii) described DNA molecular DNA molecular.

4. a kind of encoding gene of zearalenone digestive enzyme according to claim 3, it is characterised in that the strict bar Part is：Na ion concentration is in 50-300mM solution, reaction temperature is 50-68 DEG C.

5. a kind of recombinant vector, it is characterised in that include the zearalenone digestive enzyme described in any one of claim 2 to 4 Encoding gene.

6. a kind of transformant, it is characterised in that include the recombinant vector described in claim 5.

7. a kind of primer pair, it is characterised in that for expanding the zearalenone digestive enzyme described in claim any one of 2-4 Encoding gene total length and its any fragment；It is preferred that the sequence of primer pair is as shown in SEQ ID NO.3 and SEQ ID NO.4.

8. a kind of zearalenone digestive enzyme as claimed in claim 1 is in hydrolysed corn zeranol and its derivative Using.

9. application according to claim 8, it is characterised in that described derivative includes α-zearalenol, β-jade The red mould enol of rice, α-zearalanol, β-ZER.

10. a kind of method for producing zearalenone digestive enzyme, this method is including cultivating the transformant described in claim 6 simultaneously Zearalenone digestive enzyme is collected in cultured products.