Method for constructing efficient zearalenone-degrading enzyme in gastric environment of pig

Abstract

The present invention relates to the technical field of genetic engineering. Provided is a method for constructing an efficient zearalenone-degrading enzyme in the gastric environment of a pig. In the zearalenone-degrading enzyme as shown in SEQ ID NO: 1 that is taken as the parent, the amino acid at position 92, the amino acid at position 114, the amino acid at position 117, the amino acid at position 140, the amino acid at position 153, the amino acid at position 157 and the amino acid at position 220 are respectively mutated into lysine, leucine, tryptophan, arginine, methionine, valine and tryptophan to obtain the mutant. The obtained zearalenone-degrading enzyme mutant has high enzymatic activity and thermal stability, and is better adapted to the internal environment of pigs.

Description

A method for constructing an efficient enzyme for degrading zearalenone in pig stomach environment Technical Field

[0001] The present invention relates to the technical field of genetic engineering, and in particular to a method for constructing an enzyme that efficiently degrades zearalenone in a pig stomach environment. Background Art

[0002] China has a large population and high grain production. Because the humidity and temperature in most parts of China are suitable for mold growth and reproduction, mold contamination of corn, wheat, and soybeans is common. Corn, wheat, and soybeans commonly contain mycotoxins.

[0003] Some Chinese herbal medicines can become contaminated with mycotoxins during cultivation or storage due to various reasons. For example, Qichun mugwort leaves are susceptible to mold contamination during harvesting or prolonged rainy weather during storage. These contaminated molds can produce mycotoxins such as zearalenone.

[0004] Zearalenone is a type of mycotoxin that is very harmful to chickens, pigs, cattle, sheep, etc., reducing the quality of meat in the breeding industry and potentially causing harm to humans.

[0005] Enzymatic degradation of zearalenone is a green and environmentally friendly method. Enzymatic degradation of zearalenone can be performed in three scenarios: liquid degradation, solid degradation, and in the stomach of livestock and poultry. Liquid degradation, due to its low product content, requires significant energy to remove water, limiting its application. Solid degradation, due to its low water content, prevents the enzyme from fully contacting all zearalenone molecules, resulting in incomplete degradation or a very low effective degradation rate. In the stomach of livestock and poultry, the liquid form of feed allows the enzyme to fully contact zearalenone, enabling effective degradation. Furthermore, liquid feed does not require drying and concentration; it enters the intestines for direct absorption and utilization. Therefore, in-vivo degradation in livestock and poultry is an ideal method for zearalenone degradation in feed. Current enzymes that degrade zearalenone generally have high activity at around pH 6.0. However, the pH in the stomach of pigs after feeding is generally around 4.0. At this pH, existing zearalenone-degrading enzymes have low activity and cannot effectively degrade zearalenone in the stomach. Therefore, improving the activity of zearalenone-degrading enzyme at a pH of about 4.0 is of great practical significance.

[0006] Therefore, how to provide an enzyme that can efficiently degrade zearalenone in an animal's internal environment (35-37° C., pH=4.0) is a problem that those skilled in the art urgently need to solve.

[0007] Summary of the Invention

[0008] In order to address the deficiencies in the prior art, the present invention aims to provide a method for constructing an efficient zearalenone-degrading enzyme in the pig stomach environment, thereby providing a zearalenone-degrading enzyme with high enzyme activity and thermal stability, which is more adaptable to the pig's internal environment.

[0009] In order to achieve the above object, the present invention adopts the following technical solutions:

[0010] A zearalenone-degrading enzyme mutant is obtained by using the zearalenone-degrading enzyme shown in SEQ ID NO.1 as a parent, and mutating the amino acid at position 92 of the parent to lysine, the amino acid at position 114 to leucine, the amino acid at position 117 to tryptophan, the amino acid at position 140 to arginine, the amino acid at position 153 to methionine, the amino acid at position 157 to valine, and the amino acid at position 220 to tryptophan.

[0011] Preferably, the amino acid sequence of the zearalenone degrading enzyme mutant is shown in SEQ ID NO.3.

[0012] The present invention also claims a gene encoding the zearalenone degrading enzyme mutant, the gene sequence of which is shown in SEQ ID NO.4

[0013] The present invention also claims a vector containing the gene.

[0014] The present invention also claims protection for a recombinant engineered bacterium, wherein the recombinant engineered bacterium contains the vector as claimed in claim 4, or the genome contains the gene as claimed in claim 3.

[0015] The present invention also claims a method for constructing a zearalenone-degrading enzyme with high activity in the pig stomach environment, which is obtained by mutating the 92nd amino acid of the zearalenone-degrading enzyme with an amino acid sequence as shown in SEQ ID NO.1 to lysine, the 114th amino acid to leucine, the 117th amino acid to tryptophan, the 140th amino acid to arginine, the 153rd amino acid to methionine, the 157th amino acid to valine, and the 220th amino acid to tryptophan.

[0016] The present invention also claims protection for a method for constructing a zearalenone-degrading enzyme with high stability in the pig stomach environment, which is obtained by mutating the 92nd amino acid of the zearalenone-degrading enzyme shown in SEQ ID NO.1 to lysine, the 114th amino acid to leucine, the 117th amino acid to tryptophan, the 140th amino acid to arginine, the 153rd amino acid to methionine, the 157th amino acid to valine, and the 220th amino acid to tryptophan.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] The present invention provides a method for constructing an enzyme that efficiently degrades zearalenone in the pig stomach environment, obtaining a new trypsin sequence with an optimum temperature of 36°C and higher activity and thermal stability at pH = 4.0. The mutated enzyme has the ability to more efficiently degrade zearalenone in the pig stomach environment. DETAILED DESCRIPTION

[0019] In order to make the purpose, technical solutions and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with the embodiments. Of course, the specific embodiments described here are only used to explain the present invention and are not used to limit the present invention.

[0020] Unless otherwise specified, the chemical reagents and materials in the present invention are purchased from commercial sources or synthesized from commercially purchased raw materials.

[0021] The following further describes a method for improving the thermal stability of a zearalenone-degrading enzyme by combining mutants provided by the present invention. For experimental methods in the following examples where specific conditions are not specified, conventional methods and conditions were used, or as selected from the product specifications. The room temperature described herein is conventional in the art, ranging from 20°C to 40°C.

[0022] 1. Materials and instruments

[0023] (1) Strains, plasmids, and reagents

[0024] E. coli BL21 and E. coli DH5α were cultured in Luria Bertani medium (10.0 g NaCl, 10.0 g tryptone, 5.0 g yeast extract). pET-28a(+) was used to construct plasmids expressing target genes, E. coli DH5α was used as the host for plasmid cloning, and E. coli BL21 was used as the expression host strain. Zearalenone was purchased from Chengdu Pusi Biotechnology Co., Ltd.; restriction enzymes were purchased from Baoriyi Biotechnology (Beijing) Co., Ltd.; Mut II Rapid Mutation Kit V2 was purchased from Novozymes Biotech Co., Ltd.; other reagents and chemicals were purchased from Shanghai Sangon Biotech Co., Ltd. (Shanghai, China).

[0025] (2) Test equipment

[0026] Table 1 Instruments and equipment

[0027] Example 1 Vector construction of wild-type corn zearalenone-degrading enzyme

[0028] The amino acid sequence of corn zearalenone degrading enzyme (SEQ ID NO.1, the corresponding coding sequence is SEQ ID NO.2) was obtained from NCBI. According to the codon preference of E. coli, the gene sequence of corn zearalenone degrading enzyme was optimized and commissioned to a biological company for synthesis. The synthesized sequence was used as a template and amplified using primers ZHD-F / ZHD-R. After identification by agarose gel electrophoresis, the target band was recovered by gel excision. The plasmid pET-28a (+) was double-digested with EcoR I and Hind Ⅲ, and the linearized plasmid that was completely digested was excised and recovered. The recovered fragment and the linearized plasmid were ligated with ligase. The ligation product was transformed into Escherichia coli DH5α, and after colony PCR verification, it was transformed into E. coli BL21 (DE3) to express corn zearalenone degrading enzyme.

[0029] SEQ ID NO.1:

[0030] The coding sequence of the parent is shown in SEQ ID NO.2.

[0031] SEQ ID NO.2:

[0032] The ligation product was transformed into E. coli DH5α competent cells. The transformation steps are as follows:

[0033] (1) Open the constant temperature water bath in advance and adjust the temperature to 42℃.

[0034] (2) Take the competent cells DH5α / BL21(DE3) out of the -80℃ freezer in advance and place them in an ice box filled with ice to thaw naturally. After thawing, transformation can be performed.

[0035] (3) Add 2-5 μL of plasmid to an EP tube containing 100 μL of competent cells DH5α / BL21 (DE3) and place the tube in an ice bath for 20 minutes. The purpose of this step is to allow the plasmid to approach the competent cells under low temperature conditions and adsorb on the surface of the competent cells.

[0036] (4) Insert the EP tube into the foam float and place it in a 42°C water bath for 90 seconds. The purpose of this step is to change the permeability of the cell membrane of the competent cells from weak to strong during the temperature change from low temperature to 42°C, allowing the plasmid to enter the competent cells.

[0037] (5) After the heat shock is complete, immediately place the EP tube in ice and place it in an ice bath for 3 to 5 minutes.

[0038] (6) Add 800 μL of antibiotic-free LB to the EP tube and place it in a 37°C constant temperature shaker at 250 rpm for 45 minutes.

[0039] (7) Centrifuge at 12000 rpm for 1 min. Discard 700 μL of the supernatant and resuspend the competent cell pellet in the remaining 200 μL of LB. Gently pipette the resuspended bacterial solution onto a solid LB plate containing 50 μg / mL. Spread the bacterial solution evenly with a flaked spreader. Place the plate upright for 30 min until the solution is completely absorbed. Then, place the plate upside down in a 37°C incubator and culture overnight.

[0040] Single colonies grown on the LB solid medium were identified by double enzyme digestion: a single colony grown on the aforementioned solid LB medium was inoculated into 5.0 mL of liquid LB medium containing kanamycin. The culture was incubated overnight at 37°C and 200 rpm. A portion of the bacterial suspension was collected and stored in a glycerol tube. An appropriate amount of the bacterial suspension was then taken from each strain to extract the recombinant plasmid according to the instructions of the plasmid extraction kit. The extracted recombinant plasmid was double digested with EcoRI and HindIII, using the same system as for double enzyme digestion of pET-28a(+). The digested products were analyzed by 1% agarose gel electrophoresis to identify the target gene fragment and the vector fragment.

[0041] Table 2 Amplification primers for target gene zearalenone-degrading enzyme

[0042] Table 3 PCR amplification system of target gene zearalenone-degrading enzyme

[0043] Table 4 Target gene PCR reaction program

[0044] Table 5 pET28a(+) double enzyme digestion system

[0045] Example 2 Construction of mutant vector

[0046] Since all mutation sites needed to be combined for this experiment, the method adopted was to use the zearalenone-degrading enzyme with the amino acid sequence shown in SEQ ID NO.1 as the parent, first mutating the aspartic acid at position 92 to lysine, and then verifying it by sequencing. Because the amino acids at positions 114 and 117 are close to each other, continuous multi-base site-directed mutagenesis was performed. Using the plasmid with the mutated amino acid at position 92 as the template, the glycine and aspartic acid at positions 114 and 117 were mutated to leucine and tryptophan, respectively. Similarly, the valine at position 140, the valine at position 153, the aspartic acid at position 157, and the serine at position 220 were gradually mutated to arginine, methionine, valine, and tryptophan, respectively, to obtain the amino acid sequence SEQ ID NO.3.

[0047] The amino acid sequence of the mutant is shown in SEQ ID NO.3:

[0048] The coding sequence of the mutant is shown in SEQ ID NO.4:

[0049] Table 6 Mutation primer design

[0050] Single-base and multi-base mutations require direct reverse amplification of the original plasmid using reverse-complementary primers (Table 6). Multi-base mutations require designing partially reverse-complementary primers at both mutation sites A and B. The two primer pairs are interleaved to generate AB and BA segments, which are then digested with Dpn I. The AB and BA segments are mixed in appropriate proportions and reacted at 37°C for 30 minutes. Recombination is then completed under the catalysis of Exnase II, achieving in vitro circularization of the DNA. Recombinant products are then directly transformed, and positive clones formed on the plate are screened.

[0051] Table 7 Plasmid amplification reaction system

[0052] Table 8 Dpn I digested plasmid template reaction system

[0053] Table 9 Recombination reaction in vitro cyclization

[0054] Example 3 Expression of Zearalenone-degrading Enzyme

[0055] (1) Prepare seed solution: Use an inoculating loop to pick up E. coli BL21 (DE3) strain stored at -20°C in a clean bench. Streak three stripes on an LB plate containing kanamycin. Incubate the plate upside down in a 37°C incubator for 12 h. Pick a single colony from the plate and inoculate it into 50 mL of LB plate containing kanamycin.

[0056] (2) Induction culture: Add 1 mL of seed solution to LB shake flask culture medium containing kanamycin, place it at 37°C, shake it at 220 rpm, and wait until OD 600 When the pH value was 0.6, IPTG was added to a final concentration of 0.5 mM to induce protein expression in the recombinant bacteria. The temperature was lowered to 18°C ​​and the rotation speed was reduced to 120 rpm. Protein expression was induced at low temperature for 16 hours, and the induced bacteria were collected by centrifugation at 6500 rpm for 5.0 minutes. The cells were washed twice with 0.9% saline. The same procedure was performed on the recombinant bacteria without IPTG as a control.

[0057] (3) Ultrasonic disruption: Mix wet bacteria and lysis buffer (50 mM Tris-HCl, pH 8.0) in a ratio of 1:10 and stir thoroughly to fully suspend the bacteria; ultrasonically disrupt the bacteria in an ice bath under the following conditions: power 70 W, working for 20 min, running for 2 s, and stopping for 3 s; ultrasonicate twice, centrifuge at 12000 rpm and 4°C for 30 min, and collect the supernatant and precipitate respectively.

[0058] Example 4 Purification of Zearalenone-Degrading Enzyme

[0059] SDS-PAGE confirmed that the expressed zearalenone-degrading enzyme was a soluble protein.

[0060] The recombinant protein has 6× histidine at the C-terminus. The imidazole ring of histidine can bind to metal ions. 2+ Affinity chromatography columns can be used to purify the target protein. The target protein selectively binds to the nickel filler and is separated from the impurities, while high concentrations of imidazole compete with Ni. 2+ Binding, thereby achieving the effect of eluting the target protein. The specific steps of Ni-column affinity chromatography are as follows:

[0061] (1) Remove the protective ethanol in the column with 3 to 5 column volumes of distilled water;

[0062] (2) Equilibrate the column with at least 5 column volumes of binding buffer (0.5 M NaCl, 20 mM Tris-HCl, pH 8.0);

[0063] (3) Add all the supernatant to the column, and add the collected effluent to the column again to allow the target protein to fully bind to the filler;

[0064] (4) Wash the column with at least 5 column volumes of binding buffer until no yellow liquid flows out;

[0065] (5) Elution was performed using 10 to 20 column volumes of different gradient elution buffer (0.5 M NaCl, 20 mM Tris-HCl, 5 to 500 mM imidazole, pH 8.0), and the eluates were collected respectively; the collected eluates were subjected to SDS-PAGE electrophoresis, and the electrophoresis results showed that there was only one protein band.

[0066] The collected eluate was concentrated and collected using an ultrafiltration concentrator with a molecular weight cutoff of 10.0 kDa (the specifications of the ultrafiltration concentrator depend on the protein molecular weight, generally 1 / 3 of the molecular weight). The target protein was centrifuged at 4°C, 3000-3500 x g / min for 20 min to collect about 1.5 mL of the target enzyme solution. The liquid in the collection tube was discarded and replaced with desalted buffer (50 mM Tris-HCl, pH 8.0). The above centrifugation step was repeated 3-5 times. After the end, the protein in the ultrafiltration tube was aspirated into an EP tube, placed in liquid nitrogen for quick freezing for 20 s, and then immediately removed and stored at -80°C.

[0067] Example 5 Enzyme activity determination

[0068] The activity of recombinant enzyme is defined as the amount of enzyme required to consume 1 μg of substrate per unit time is 1 U.

[0069] The total enzyme reaction volume was 500 μL, containing: 10 μL enzyme solution (concentration 0.5 mg / mL), 10 μL substrate ZEN (concentration 1 mg / mL), and 480 μL buffer (buffer pH 4.0, 10.12 g potassium hydrogen phthalate dissolved in water and diluted to 1 L in a volumetric flask). The enzyme reaction system was incubated at 37°C for 10 minutes and immediately inactivated with 500 μL of methanol. The reaction was then incubated on ice for 10 minutes, filtered through a 0.22 μm organic filter membrane, and enzyme activity was determined by HPLC.

[0070] The HPLC detection conditions were as follows: the detector was a fluorescence detector, the excitation wavelength was 274 nm, the emission wavelength was 440 nm, the chromatographic column was a WondaSil-C18 column (150×4.6 mmol / L, 5 μm), the mobile phase was acetonitrile:water = 5:5, the flow rate was 1 mL / min, and the column temperature was 30°C.

[0071] Preparation of standard curve: 1 mg / mL ZEN stock solution was diluted to 0.5, 1, 2, 4, 8, 16, and 32 μg / mL and filtered through a 0.22 μm filter membrane.

[0072] Example 6 Determination of the Thermal Stability and Optimum Temperature of Zearalenone-Degrading Enzyme

[0073] Zearalenone-degrading enzyme was incubated at 50°C for 0 min, 2 min, 5 min, 7 min, and 10 min, and then ice-bathed for 60 s before measuring enzyme activity. The activity before incubation was set as 100%. The enzyme inactivation half-life was calculated according to the reference (L. Liu, H. Yu, K. Du, Z. Wang, Y. Gan, H. Huang, Enhanced trypsin thermostability in Pichia pastoris through truncating the flexible region, Microb. Cell Factor. 17(1)(2018)165, https: / / doi.org / 10.1186 / s12934-018-1012-x).

[0074] Determination of the optimal temperature of zearalenone-degrading enzyme: The temperature at which the zearalenone-degrading enzyme has the highest activity is 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, and 40°C. This is the optimal temperature of the enzyme.

[0075] Determination of the optimal pH for zearalenone-degrading enzyme: Sodium citrate buffers were prepared at pH 4.0, pH 5.0, pH 6.0, and pH 7.0. A 0.2M Na2HPO4 aqueous solution and a 0.1M sodium citrate aqueous solution were mixed at a volume ratio of 7.71:12.29 to obtain a pH 4.0 enzyme reaction buffer. A 0.2M Na2HPO4 aqueous solution and a 0.1M sodium citrate aqueous solution were mixed at a volume ratio of 10.30:9.70 to obtain a pH 5.0 enzyme reaction buffer. A 0.2M Na2HPO4 aqueous solution and a 0.1M sodium citrate aqueous solution were mixed at a volume ratio of 12.63:7.37 to obtain a pH 6.0 enzyme reaction buffer. A 0.2M Na2HPO4 aqueous solution and a 0.1M sodium citrate aqueous solution were mixed at a volume ratio of 16.47:3.53 to obtain a pH 7.0 enzyme reaction buffer.

[0076] Different enzyme reaction buffers were used to prepare reaction systems of different enzymes. The specific enzyme activities were measured in the different enzyme reaction systems. The pH with the maximum specific enzyme activity was defined as the optimal pH.

[0077] The specific enzyme activity of the mutant corn zearalenone-degrading enzyme was measured to be 232.99 U / mg, the half-life was measured to be 15.0 min, the optimal temperature was measured to be 36°C, and the optimal pH was measured to be 4.0.

[0078] Comparative Example 1

[0079] The initial sequence (amino acid sequence is SEQ ID NO.1, base sequence is SEQ ID NO.2) was expressed and purified as in Example 1. The specific enzyme activity was measured to be 160.03 U / mg, the half-life was measured to be 2.8 min, and the optimum temperature was measured to be 37°C.

[0080] The above description is only a preferred specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any technician familiar with the technical field, within the technical scope disclosed by the present invention, who makes equivalent replacements or changes based on the technical solution and inventive concept of the present invention, should be covered by the scope of protection of the present invention.

Claims

1. A zearalenone-degrading enzyme mutant, characterized in that: The mutant is obtained by using the zearalenone-degrading enzyme shown in SEQ ID NO.1 as a parent, and mutating the 92nd amino acid of the parent to lysine, the 114th amino acid to leucine, the 117th amino acid to tryptophan, the 140th amino acid to arginine, the 153rd amino acid to methionine, the 157th amino acid to valine, and the 220th amino acid to tryptophan.

2. The zearalenone-degrading enzyme mutant according to claim 1, characterized in that The amino acid sequence of the zearalenone degrading enzyme mutant is shown in SEQ ID NO.

3.

3. A gene encoding the zearalenone-degrading enzyme mutant according to claim 1, characterized in that: The gene sequence is shown in SEQ ID NO.

4.

4. A carrier, characterized in that The vector contains the gene according to claim 3.

5. A recombinant engineered bacterium, characterized in that: The recombinant engineered bacteria contains the vector according to claim 4, or the genome contains the gene according to claim 3.

6. A method for constructing a zearalenone-degrading enzyme with high activity in the pig stomach environment, characterized in that: The zearalenone degrading enzyme is obtained by mutating the amino acid at position 92 to lysine, the amino acid at position 114 to leucine, the amino acid at position 117 to tryptophan, the amino acid at position 140 to arginine, the amino acid at position 153 to methionine, the amino acid at position 157 to valine, and the amino acid at position 220 to tryptophan.

7. A method for constructing a zearalenone-degrading enzyme with high stability in pig stomach environment, characterized in that: The zearalenone degrading enzyme is obtained by mutating the amino acid at position 92 to lysine, the amino acid at position 114 to leucine, the amino acid at position 117 to tryptophan, the amino acid at position 140 to arginine, the amino acid at position 153 to methionine, the amino acid at position 157 to valine, and the amino acid at position 220 to tryptophan.

Images