A zearalenone-degrading enzyme mutant, and a preparation method and application thereof

Abstract

The present application relates to the technical field of enzyme engineering, and particularly relates to a zearalenone degrading enzyme mutant, a preparation method and application thereof, wherein the amino acid sequence of the zearalenone degrading enzyme mutant is shown as SEQ ID NO: 4. The zearalenone degrading enzyme mutant can efficiently degrade zearalenone in a short time, and can be used in various corn by-products. Compared with the wild-type zearalenone degrading enzyme, the zearalenone degrading enzyme mutant of the present application exhibits more excellent thermal stability, can be more effectively detoxified at high temperature, and better meets the requirements of industrial application.

Description

Technical Field

[0001] This invention relates to the field of enzyme engineering technology, specifically to a zearalenone-degrading enzyme mutant, its preparation method, and its application. Background Technology

[0002] Corn is susceptible to mold contamination during planting, harvesting, storage, and processing, especially in humid environments where it is more easily contaminated with one or more toxins. After mold infection, suitable temperature and humidity conditions during processing and storage of corn and its byproducts promote the formation of zearalenone. Furthermore, with deeper processing of corn, toxins accumulate in byproducts along with nutrients. In some regions, high detection rates of vomitoxin and zearalenone (ZEN) have been found in feed from corn and corn byproducts, with the detection rate in byproducts being higher than in raw materials. Therefore, excessive zearalenone content in corn byproducts is more common.

[0003] Several countries around the world have established limits for ZEN in corn, food, and vegetable oils. The European Union standard (EC1126-2007) sets limits for ZEN in cereals, corn, and cereal products, specifying that the ZEN content in refined corn oil cannot exceed 400 μg / kg. Austria stipulates that the ZEN content in wheat, rye, and durum wheat must not exceed 60 μg / kg. Russia stipulates that the ZEN content in durum wheat and flour cannot exceed 1000 μg / kg. France stipulates that the ZEN content in cereals must be less than 200 μg / kg. my country's food standard GB 2761-2011 explicitly stipulates that the ZEN content in wheat, wheat flour, corn, and cornmeal must not exceed 60 μg / kg. The feed hygiene standard GB 13078.2-2006 stipulates that the ZEN content in feed corn and compound feed must not exceed 500 μg / kg.

[0004] Furthermore, by-products can become contaminated with ZEN during processing, storage, and other product distribution processes, and ZEN can accumulate in animals through the food chain, ultimately affecting human health. To reduce or even eliminate the impact of ZEN-contaminated grain products and animal feed on humans and animals, there is an urgent need to find an excellent ZEN degradation method.

[0005] Currently, methods for removing zearalenone and its derivatives include physical, chemical, and biological methods. Physical methods use high-intensity heating, irradiation, and adsorption to reduce zearalenone, but often fail to completely remove it. Chemical methods, including alkaline hydrolysis, ozone oxidation, and ammoniation, are unsuitable for practical production applications due to their high risk, low efficiency, and tendency to cause secondary pollution. Compared to the limitations of these two detoxification methods, biological degradation methods utilize microbial cells to degrade toxins, and secondary metabolites or enzymes produced by microorganisms during their growth and metabolism specifically degrade zearalenone under mild conditions, forming less toxic or non-toxic degradation products. Biological detoxification methods can be scaled up without damaging the nutrients in feed, and possess strong specificity and particularity. They are highly efficient, environmentally friendly, and avoid secondary pollution, making them significantly advantageous in zearalenone degradation and considered the best method for food and feed detoxification.

[0006] Recently, various microorganisms and enzymes with the ability to detoxify or attenuate zearalenone have been identified and explored, and a considerable number of probiotics show promising application prospects as feed additives. Therefore, utilizing non-pathogenic microorganisms to degrade zearalenone and developing highly efficient degrading enzymes are important means to solve the problem of zearalenone pollution. However, most of the screened zearalenone-degrading enzymes have not yet been industrialized, and the quality of detoxifying agents on the market varies greatly. Some products have low effective content, resulting in the inability to reduce the toxin content to a safe level and poor detoxification effect. Summary of the Invention

[0007] The purpose of this invention is to provide a zearalenone-degrading enzyme mutant, its preparation method, and its application.

[0008] To achieve the above objectives, the present invention provides the following technical solution: A mutant zearalenone-degrading enzyme has the amino acid sequence shown in SEQ ID NO: 4 and the nucleotide sequence shown in SEQ ID NO: 3. Correspondingly, the nucleotide and amino acid sequences of the wild-type zearalenone-degrading enzyme are shown in SEQ ID NO: 1 and SEQ ID NO: 2.

[0009] The method for preparing the zearalenone-degrading enzyme mutant of this invention specifically comprises: taking an engineered Escherichia coli strain containing a plasmid, inoculating it into 50 mL of LB medium containing 100 μg / mL ampicillin, and incubating at 37°C with shaking at 200 r / min until OD200. 600 Value 0.6-0.8, 1% was transferred to 100 mL of LB medium containing 100 μg / mL ampicillin, and cultured at 37℃ with shaking at 200 rpm until OD. 600With a pH of 0.6-0.8, add 0.2 mM IPTG and induce at 20-25℃ for 10 h. Collect the bacterial cells by centrifugation, resuspend the bacterial cells in a pH 6.5 phosphate buffer solution, sonicate to disrupt, filter the disrupted liquid through a 0.45 μm aqueous membrane to obtain the enzyme solution, add maltodextrin, and spray dry to prepare enzyme powder for later use.

[0010] The final enzyme activity of the prepared enzyme powder is 2000 U / g.

[0011] The zearalenone-degrading enzyme mutant described in this invention can be used to reduce toxins in maize by-products.

[0012] The corn by-products include corn steep liquor, corn gluten meal aqueous solution, and corn oil.

[0013] When used to degrade ZEN in corn steep liquor, the temperature is 40℃, the ratio of corn steep liquor to water is 1:2, and the amount of enzyme powder added is ≥1000g / t.

[0014] When used to degrade ZEN in an aqueous solution of corn gluten meal, the pH of the aqueous solution of corn gluten meal should be between 6.5 and 7.5, the ratio of corn gluten meal to water should be 1:10, and the amount of enzyme added should be ≥750g / t.

[0015] When used to degrade ZEN in corn oil, the ratio of corn oil to water is 1:0.1, and the amount of enzyme powder added is ≥800g / t.

[0016] Compared with the prior art, the beneficial effects of the present invention are: This invention provides a zearalenone-degrading enzyme mutant that can degrade zearalenone efficiently in a short time and can be used in various maize by-products. Compared with wild-type zearalenone-degrading enzyme, the zearalenone-degrading enzyme mutant of this invention exhibits superior thermal stability, can more effectively detoxify at high temperatures, and better meets the requirements of industrial applications. Attached Figure Description

[0017] Figure 1 A map was constructed for the pET-22b(+)-zeng vector. The map includes: T7 promoter (19 bp), T7 terminator (48 bp), Lac operator (used to control the transcriptional activity of T7 RNA polymerase), Lac I (lactose repressor protein), AmpR (861 bp ampicillin resistance gene, providing a selection marker for recombinant E. coli strains), AmpR promoter (105 bp ampicillin gene promoter), and Ori (plasmid replication initiation site). Detailed Implementation

[0018] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0019] Example 1 Preparation of zearalenone-degrading enzyme Wild-type carbonyl reductase and mutant sequences were synthesized and then amplified by PCR (DNA sequences are shown in SEQ ID NO: 1 and 3). The recombinant expression vector was then introduced into the NdeI and XhoI restriction sites of the expression vector pET-22b(+) to obtain the recombinant expression vector. The construction map of the pET-22b(+)-zeng vector is shown below. Figure 1 As shown. The strain was transformed into Escherichia coli BL21(DE3), and cloned strains were obtained after screening using antibiotic resistance plates. The specific method is as follows: I. Mutant Target Based on molecular dynamics simulations, the root mean square fluctuation (RMSF) of each amino acid residue in the ZENG protein was calculated to screen sites with high structural flexibility and significant impact on thermal stability. Ultimately, glutamine at position 37 (Q37), leucine at position 155 (L155), and histidine at position 248 (H248) were identified as key modification targets.

[0020] Site-directed mutagenesis was employed to replace Q37 with threonine (T) to enhance the number of local hydrogen bonds; L155 with proline (P) to improve α-helix rigidity; and H248 with glutamate (E) to optimize local electrostatic interactions. This experiment constructed single-point mutants Q37T, L155P, and H248E, as well as a three-point combination mutant Q37T / L155P / H248E, to achieve a synergistic improvement in enzyme thermostability.

[0021] II. Primer Design Based on the amino acid substitution requirements at the mutation sites, site-directed mutagenesis primers were designed using whole-plasmid PCR. To cover all amino acid types, saturation mutagenesis primers containing the NNK degenerate codon were also designed. The primers were synthesized by a biotechnology company, and their sequences are shown in the table below.

[0022] ① Site-directed mutagenesis primers (sequences shown in SEQ ID NO: 5-10) ② Saturation mutation primers (containing NNK degenerate codons, sequences shown in SEQ ID NO: 11-16) III. Construction of mutant plasmids (construction map of pET-22b(+)-zeng vector as shown in the figure) Figure 1 (As shown) 1. PCR amplification Using the pET-22b(+) recombinant plasmid carrying the wild-type ZENG gene as a template, PCR amplification was performed using the primers described above. The reaction mixture (25 μL) consisted of 12.5 μL of 2× PrimeSTAR Max Premix, 1 μL each of forward and reverse primers (10 μM), 0.5 μL of template plasmid, and ddH2O to a final volume of 25 μL. PCR conditions were as follows: 95℃ pre-denaturation for 3 min; 94℃ denaturation for 30 s, 55℃ annealing for 40 s, 70℃ extension for 4 min, for a total of 30 cycles; and a final extension at 72℃ for 5 min. The mixture was stored at 4℃.

[0023] 2. Template digestion and transformation After the PCR products were verified by 1% agarose gel electrophoresis, 1 μL of QuickCut DpnI and the corresponding buffer were added to the reaction solution and incubated at 40℃ for 1 h to eliminate the original methylated template plasmid.

[0024] 3. Conversion and Screening The digested PCR product was transformed into E. coli JM109 competent cells and plated on LB agar plates containing ampicillin (50 μg / mL), and incubated overnight at 37°C. Single colonies were picked, plasmids were extracted, and mutation sites were confirmed by Sanger sequencing. The correctly sequenced mutant plasmid was transformed into the expression host E. coli BL21(DE3) for subsequent protein expression and functional validation.

[0025] 4. Construction of combined mutants A point-by-point superposition mutation strategy was adopted to construct three key combination mutants: using the wild-type plasmid as a template, a Q37T single-point mutant was constructed; using the Q37T mutant plasmid as a template, a Q37T / L155P double-point mutant was constructed; and using the Q37T / L155P double-mutant plasmid as a template, a Q37T / L155P / H248E three key combination mutant was constructed.

[0026] Each round of mutations was verified by PCR amplification, DpnI digestion, transformation, and Sanger sequencing to ensure the accuracy of the mutation sites.

[0027] IV. Mutant Expression The engineered strain of *E. coli* containing the plasmid was inoculated into 50 mL of LB medium containing 100 μg / mL ampicillin and cultured at 37°C with shaking at 200 rpm until OD200. 600Value 0.6-0.8, 1% was transferred to 100 mL of LB medium containing 100 μg / mL ampicillin, and cultured at 37℃ with shaking at 200 rpm until OD. 600 With a pH of 0.6-0.8, add 0.2 mM IPTG and induce at 20-25℃ for 10 h. Collect the bacterial cells by centrifugation, resuspend the bacterial cells in a pH 6.5 phosphate buffer solution, sonicate to disrupt, filter the disrupted liquid through a 0.45 μm aqueous membrane to obtain the enzyme solution, add maltodextrin, and spray dry to prepare enzyme powder for later use (final enzyme activity is 2000 U / g).

[0028] Example 2: Determination of the thermal stability of zearalenone-degrading enzymes The zearalenone-degrading enzyme mutant was incubated at 55℃ for 2, 4, and 6 min, then immediately placed in an ice bath, and the enzyme reaction was carried out according to the standard enzyme reaction system and conditions in the enzyme activity assay. The initial enzyme activity before incubation was defined as 100%, and the relative residual enzyme activity (%) was defined as the ratio of residual activity to initial enzyme activity.

[0029] Enzyme activity detection methods: (1) Enzyme reaction assay system: The total volume of the system is 1 mL, including 20 μL of pure enzyme solution, 10 μL of substrate ZEN (5 g / L), and 970 μL of phosphate buffer (pH 6.5).

[0030] (2) Enzyme reaction assay conditions: Place the above enzyme reaction system in a constant temperature water bath, time accurately, and after the reaction is completed, quickly add 1 mL of methanol for inactivation treatment. Take 1 mL of supernatant and pass it through a membrane for HPLC detection.

[0031] As shown in the table above, the relative residual enzyme activity of the zearalenone-degrading enzyme mutant showed only a slight decrease after incubation at 55℃ for 2 min. After incubation for 6 min, it retained 90% of the relative residual enzyme activity under the same conditions.

[0032] The thermostability of wild-type zearalenone-degrading enzyme was determined using the same method, and it was found that the wild-type zearalenone-degrading enzyme essentially lost its activity after incubation at 53°C for 2 minutes. Therefore, the zearalenone-degrading enzyme mutant of this invention exhibits superior thermostability, can be more effectively detoxified at high temperatures, and better meets the requirements of industrial applications.

[0033] Example 3: Application of zearalenone-degrading enzyme mutant in degrading toxins in maize by-products This invention detects the efficiency of zearalenone-degrading enzymes in degrading ZEN in corn steep liquor, corn gluten meal, and corn oil. The specific implementation steps are as follows: 1. Zearalenone-degrading enzymes degrade ZEN in corn steep liquor A. The effect of temperature on the efficiency of ZEN degradation by enzyme powder Corn steep liquor was divided into four groups: a control group (Group C) and three experimental groups (Group T), with three replicates per group. Each Group T received 1000 g / t of enzyme powder and was incubated with shaking at 200 rpm for 2 hours. The reaction temperatures were 35℃ for Group T1, 40℃ for Group T2, and 45℃ for Group T3. Toxin levels were immediately detected using ZEN test strips after the reaction.

[0034] The results are shown in the table below. At a temperature of 40℃, the enzyme powder showed the highest degradation efficiency of ZEN in corn steep liquor.

[0035] B. The effect of different ratios of corn steep liquor and water mixtures on the efficiency of ZEN degradation by enzyme powder. Take an appropriate amount of corn steep liquor and mix it with water in ratios of 1:1, 1:2, and 1:3 respectively. Add different proportions of alkaline solution to adjust the pH of the solution to 6.5-7.5. Divide each mixture into two parts: the control group (Group C) is a blank mixture, and the experimental group (Group T) is a mixture with 1000g / t enzyme powder added. Each group has 3 replicates. The reaction conditions are 40℃, shaking culture at 200r / min for 2h. After the reaction, the toxin content is immediately detected using ZEN test strips.

[0036] The results are shown in the table below. When the ratio of corn steep liquor to water is 1:2, the enzyme powder has the highest degradation efficiency for ZEN in corn steep liquor.

[0037] ① When corn steep liquor and water are mixed in a 1:1 ratio, the ZEN degradation results are as follows. ② When corn steep liquor and water are mixed in a 1:2 ratio, the ZEN degradation results are as follows. ③ When corn steep liquor and water are mixed in a 1:3 ratio, the ZEN degradation results are as follows. C. Effect of different amounts of zearalenone-degrading enzyme added on ZEN degradation efficiency Take an appropriate amount of corn steep liquor and mix it with water in a 1:2 ratio. Add an appropriate amount of alkali solution to adjust the pH of the solution to 6.5-7. Divide the mixture into 4 groups: a control group (C group) and 3 experimental groups (T groups), with 3 replicates per group. Add 250g / t enzyme powder to T1 group, 500g / t enzyme powder to T2 group, and 1000g / t enzyme powder to T3 group. The reaction conditions are 40℃, shaking and incubating at 200r / min for 2h. After the reaction is completed, immediately use ZEN test strips to detect the toxin content.

[0038] The results are shown in the table below. The degradation efficiency of ZEN increases with the increase of enzyme powder content. When the amount of enzyme powder added is 1000g / t, the degradation efficiency exceeds 80%.

[0039] 2. Zearalenone-degrading enzyme degrades ZEN in corn gluten meal. A. The effect of corn gluten meal aqueous solutions with different pH values ​​on the efficiency of ZEN degradation by enzyme powder. Take an appropriate amount of corn gluten powder and mix it with water at a ratio of 1:10. Use a high-speed blender to fully crush and mix it evenly. Divide the mixture into 4 groups: a control group (C group) and 3 experimental groups (T groups), with 3 replicates for each group. Adjust the pH of T1 group to 5-6, T2 group to 6.5-7.5, and T3 group to 8-9. Add 500g / t of enzyme powder to each experimental group. The reaction conditions are 40℃, 200r / min shaking culture for 2h. After the reaction, immediately use ZEN test strips to detect the toxin content.

[0040] The results are shown in the table below. When the pH of the aqueous solution of corn gluten meal is between 6.5 and 7.5, the enzyme powder has the highest degradation efficiency for ZEN in corn gluten meal.

[0041] B. The effect of different ratios of corn gluten meal and water mixtures on the efficiency of ZEN degradation by enzyme powder. Take an appropriate amount of corn gluten powder and mix it with water at ratios of 1:5, 1:10, and 1:15 respectively. Use a high-speed blender to fully crush and mix the mixture evenly. Add different proportions of alkaline solution to adjust the pH of the solution to 6.5-7.5. Divide each mixture into two parts: the control group is a blank mixture and the experimental group is a mixture with 500g / t enzyme powder added. Each group has 3 replicates. The reaction conditions are 40℃, shaking and incubating at 200r / min for 2h. After the reaction, immediately use ZEN test strips to detect the toxin content.

[0042] The results are shown in the table below. When the ratio of corn gluten meal to water is 1:10, the enzyme powder has the highest degradation efficiency for ZEN in corn gluten meal.

[0043] ① When corn gluten meal and water are mixed in a 1:5 ratio, the ZEN degradation results are as follows. ② Mix corn gluten meal and water in a 1:10 ratio. ZEN degradation results. ③ Mix corn gluten meal and water at a ratio of 1:15. ZEN degradation results. C. Effect of different amounts of zearalenone-degrading enzyme added on ZEN degradation efficiency Mix an appropriate amount of corn gluten powder with water at a ratio of 1:10, and thoroughly crush and mix the mixture using a high-speed blender. Add an appropriate amount of alkaline solution to adjust the pH of the solution to 6.5-7.5. Divide the mixture into 4 groups: a control group (C group) and 3 experimental groups (T groups), with 3 replicates per group. Add 250g / t enzyme powder to T1 group, 500g / t enzyme powder to T2 group, and 750g / t enzyme powder to T3 group. The reaction conditions are 40℃, shaking and incubating at 200r / min for 2h. After the reaction, immediately use ZEN test strips to detect the toxin content.

[0044] The results are shown in the table below. The degradation efficiency of ZEN increases with the increase of enzyme powder content. When the amount of enzyme powder added is 750g / t, the degradation efficiency exceeds 80%.

[0045] 3. Zearalenone-degrading enzymes degrade ZEN in corn oil A. The effect of different ratios of corn oil and water mixtures on the efficiency of ZEN degradation by enzyme powder. Take an appropriate amount of corn oil and mix it with water at ratios of 1:0, 1:0.1, and 1:0.2 respectively. Use a homogenizer to fully mix the oil and water to emulsify them. Add different proportions of alkali solution to adjust the pH of the solution to 6.5-7.5. Divide each emulsion into two parts: the control group (Group C) is a blank emulsion, and the experimental group (Group T) is an emulsion with 500g / t enzyme powder added. Each group has 3 replicates. The reaction conditions are 40℃, shaking culture at 200r / min for 2h. After the reaction, immediately use ZEN test strips to detect the toxin content.

[0046] The results are shown in the table below. When the ratio of corn oil to water is 1:0.1, the enzyme powder has the highest degradation efficiency for ZEN in corn oil.

[0047] ① When corn oil and water are mixed in a 1:0 ratio, the ZEN degradation results are as follows. ② When corn oil and water are mixed in a 1:0.1 ratio, the ZEN degradation results are as follows. ③ When corn oil and water are mixed in a 1:0.2 ratio, the ZEN degradation results are as follows. B. Effect of different amounts of zearalenone-degrading enzyme added on ZEN degradation efficiency Mix an appropriate amount of corn oil and water at a ratio of 1:0.1, and homogenize the mixture thoroughly using a homogenizer to emulsify the oil and water. Add an appropriate amount of alkali solution to adjust the pH of the solution to 6.5-7.5. Divide the mixture into 4 groups: a control group (C group) and 3 experimental groups (T groups), with 3 replicates per group. Add 250g / t enzyme powder to T1 group, 500g / t enzyme powder to T2 group, and 800g / t enzyme powder to T3 group. The reaction conditions are 40℃, shaking and incubating at 200r / min for 2h. After the reaction, immediately use ZEN test strips to detect the toxin content.

[0048] The results are shown in the table below. The degradation efficiency of ZEN increases with the increase of enzyme powder content. When the amount of enzyme powder added is 800g / t, the degradation efficiency exceeds 80%.

[0049] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A mutant of zearalenone-degrading enzyme, characterized in that: Its amino acid sequence is shown in SEQ ID NO:

4.

2. The method for preparing the zearalenone-degrading enzyme mutant according to claim 1, characterized in that: The engineered strain of *E. coli* containing the plasmid was inoculated into 50 mL of LB medium containing 100 μg / mL ampicillin and cultured at 37°C with shaking at 200 rpm until OD500. 600 Value 0.6-0.8, 1% was transferred to 100 mL of LB medium containing 100 μg / mL ampicillin, and cultured at 37℃ with shaking at 200 rpm until OD. 600 With a pH of 0.6-0.8, add 0.2 mM IPTG and induce at 20-25℃ for 10 h. Collect the bacterial cells by centrifugation, resuspend the bacterial cells in a pH 6.5 phosphate buffer solution, sonicate to disrupt, filter the disrupted liquid through a 0.45 μm aqueous membrane to obtain the enzyme solution, add maltodextrin, and spray dry to prepare enzyme powder for later use.

3. The method for preparing the zearalenone-degrading enzyme mutant according to claim 2, characterized in that: The final enzyme activity of the prepared enzyme powder is 2000 U / g.

4. The application of the zearalenone-degrading enzyme mutant of claim 1 in reducing the toxins in maize by-products.

5. The application of the zearalenone-degrading enzyme mutant according to claim 4 in reducing toxins in maize by-products, characterized in that: The corn by-products include corn steep liquor, corn gluten meal aqueous solution, and corn oil.

6. The application of the zearalenone-degrading enzyme mutant according to claim 5 in reducing toxins in maize by-products, characterized in that: When used to degrade ZEN in corn steep liquor, the temperature is 40℃, the ratio of corn steep liquor to water is 1:2, and the amount of enzyme powder added is ≥1000g / t.

7. The application of the zearalenone-degrading enzyme mutant according to claim 5 in reducing toxins in maize by-products, characterized in that: When used to degrade ZEN in an aqueous solution of corn gluten meal, the pH of the aqueous solution of corn gluten meal should be between 6.5 and 7.5, the ratio of corn gluten meal to water should be 1:10, and the amount of enzyme added should be ≥750g / t.

8. The application of the zearalenone-degrading enzyme mutant according to claim 5 in reducing toxins in maize by-products, characterized in that: When used to degrade ZEN in corn oil, the ratio of corn oil to water is 1:0.1, and the amount of enzyme powder added is ≥800g / t.

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