Application of a Fusarium graminearum deoxynivalenol-degrading fusion enzyme in corn by-products

Abstract

The present invention discloses the application of a zearalenone-degrading fusion enzyme in corn by-products, belonging to the fields of microorganisms and food safety. The present invention provides a zearalenone lactonohydrolase fusion enzyme S1v1-GS-ZHD518, and the activity, expression level and thermal stability of ZHD518 lactonase are improved by fusing the S1v1 peptide. The present invention applies the fusion enzyme S1v1-GS-ZHD518 to degrade zearalenone in positive samples. This enzyme can adapt to the high-temperature conditions in the processing environment and can efficiently degrade zearalenone. The degradation rates of ZEN in corn bran, corn germ meal and corn protein powder within 1 h are 90.975%, 91.004% and 95.32% respectively, showing great application prospects.

Description

Technical Field

[0001] The present invention relates to the application of a fusion enzyme capable of degrading zearalenone in corn by-products, belonging to the fields of microbiology and food safety. Background Art

[0002] Zearalenone (ZEN), also known as F-2 toxin, is a non-steroidal estrogen mycotoxin initially discovered in moldy corn and mainly distributed in corn and grains. ZEN is biosynthesized by some common soil fungi, such as Fusarium graminearum (also known as Gibberella zeae), Fusarium cerealis, Fusarium martii, Fusarium curvulum, and Fusarium semitectum. Generally, ZEN appears as white granules, has excellent thermal stability, and is soluble in chloroform, alcohols, and some alkaline solutions. Therefore, ZEN is insensitive to environmental temperature changes and is difficult to degrade during the processing and storage of food or feed. For example, corn grains are contaminated by mycotoxins to varying degrees during growth, harvesting, and storage, and mycotoxins will disperse into various by-products during the processing. By-products such as corn steep liquor, germ meal, corn gluten meal, and corn bran feed can be produced during the processing of corn. These by-products are characterized by low price and rich nutrition and have great utilization potential, and are usually used as animal feed. The mycotoxin residues in feed raw materials will seriously endanger the growth performance and immune ability of animals. In addition, due to its structure being similar to that of 17-β-estradiol and other natural estrogens, ZEN can competitively bind to estrogen receptors with natural estrogens in the body, exert estrogen-like effects, disrupt the estrogen level in the body, and cause reproductive system diseases. In addition, ZEN is known to have immunotoxicity, hepatotoxicity, hematotoxicity, genotoxicity, teratogenicity, and carcinogenicity. Therefore, solving the pollution caused by ZEN has become one of the most concerned issues in the food and feed industries.

[0003] The degradation of mycotoxins in grains is usually achieved by physical, chemical, and biological methods. Compared with physical and chemical methods, biocatalytic digestion is more targeted, convenient, and environmentally friendly. Biocatalytic detoxification involves the use of key genes responsible for the degradation of microbial toxins. Through the application of genetic engineering, optimized microbial populations capable of highly expressing these biocatalysts are cultivated, and then pure enzymes are isolated to eliminate mycotoxins in food and feed. Among the ZEN detoxifying enzymes, lactonohydrolase is the most intensively studied and widely used enzyme. ZHD518 lactonohydrolase is a neutral ZEN-degrading enzyme. Compared with other lactones, it does not require a highly alkaline environment to achieve optimal enzyme activity, making it a better candidate for ZEN-degrading enzymes in the food industry. However, the main challenges faced by ZHD518 and other newly discovered enzymes are their low degradation efficiency and reduced stability at high temperatures. This not only affects the degradation ability of lactonase in actual positive samples but also greatly affects its compatibility with existing food and feed processing steps. Summary of the Invention

[0004] The present invention provides a fusion enzyme of zearalenone degrading enzyme, which has a polypeptide sequence with the structure shown as S1v1-GS-ZHD518; wherein, S1v1 is AEAEAHAHAEAEAHAH; GS is a GS linker peptide; ZHD518 is a zearalenone degrading enzyme with an amino acid sequence as shown in GenBank accession number: XM_013418296.1.

[0005] In one embodiment, the amino acid sequence of the fusion enzyme is as shown in SEQ ID NO.1.

[0006] The present invention also provides a gene encoding the fusion enzyme.

[0007] In one embodiment, the nucleotide sequence of the gene is as shown in SEQ ID NO.2.

[0008] The present invention also provides a recombinant Escherichia coli producing the fusion enzyme.

[0009] In one embodiment, the recombinant Escherichia coli uses pET22b as an expression vector to express the fusion enzyme shown in SEQ ID NO.1.

[0010] The present invention also provides a method for preparing the fusion enzyme, which includes: culturing the recombinant Escherichia coli and collecting the fusion enzyme in the cell culture solution.

[0011] In one embodiment, the method is to culture the recombinant Escherichia coli in LB medium for a period of time, induce with IPTG, and collect the fusion enzyme in the bacterial cells.

[0012] In one embodiment, the method further includes cell disruption, collecting the enzyme in the supernatant, and purification steps.

[0013] In one embodiment, the induction is when the culture reaches an OD of the bacterial liquid 600 = 0.5 - 0.7, adding the inducer IPTG to a final concentration of 0.1 - 0.5 mM, and inducing at 14 - 18 °C and 100 - 200 rpm for 12 - 36 h.

[0014] The present invention also provides the application of the fusion enzyme S1v1-GS-ZHD518 of the zearalenone degrading enzyme in zearalenone degradation.

[0015] In one embodiment, the application is to degrade zearalenone in corn by-products.

[0016] In one embodiment, the corn by-products include but are not limited to corn husk, corn germ meal, or corn protein powder.

[0017] In one embodiment, the reaction pH is 3.0 - 10.6.

[0018] In one embodiment, the reaction temperature is 25 - 60 °C.

[0019] In one embodiment, the reaction temperature and reaction time are 40, 45, 50, 55, 60 °C and 5, 10, 15, 30 min.

[0020] In one embodiment, the application specifically includes: corn germ meal, corn bran, and corn protein powder obtained from a small - scale corn processing workshop are ground and evenly screened through a 40 - mesh sieve. The PBS solution containing the target protein is placed in a freeze - dryer and dried overnight to obtain solid target protein. When enzymatically degrading ZEN, 200 μg of the purified S1v1 - GS - ZHD518 enzyme is added to a mixture of 5 g of corn by - products and 20 mL of PBS (pH 7.5) for reaction.

[0021] In one embodiment, the corn germ meal, corn bran, and corn protein powder are crushed and then screened through a 40 - mesh sieve.

[0022] In one embodiment, the S1v1 - GS - ZHD518 enzyme is a freeze - dried solid enzyme preparation.

[0023] In one embodiment, the reaction temperature is 45 °C.

[0024] In one embodiment, the reaction time is 15, 30, 60 min.

[0025] The present invention also provides the application of the fusion enzyme of the zearalenone - degrading enzyme in the field of food or feed processing.

[0026] Beneficial effects:

[0027] The present invention provides a recombinant enzyme S1v1 - GS - ZHD518 containing zearalenone lactonohydrolase ZHD518, which can further improve the activity, expression level, and thermal stability of the ZHD518 enzyme. Compared with the original enzyme ZHD518, the protein expression level of the recombinant enzyme S1v1 - GS - ZHD518 is increased by 1.28 times, the enzyme activity is increased by 9.27 times, and the thermal stability at 45 °C for 10 min is increased by 37.08 times. It has great application prospects.

[0028] The present invention also provides a method for efficiently degrading zearalenone in corn by - products such as corn bran, corn germ meal, and corn protein powder. With specific degradation conditions, the removal rates of zearalenone can reach 90.975%, 91.004%, and 95.32% respectively. Brief Description of the Drawings

[0029] Figure 1 It is the SDS-PAGE electrophoresis result of the recombinant enzyme S1v1-GS-ZHD518. M: marker; 1: the broken supernatant passed through the nickel column; 2-9: purified S1v1-GS-ZHD518 (33.9KDa), and 8 tubes were sequentially collected according to the different elution times of the target protein.

[0030] Figure 2 It is the optimal pH graph of the recombinant enzyme S1v1-GS-ZHD518;

[0031] Figure 3 It is the optimal temperature graph of the recombinant enzyme S1v1-GS-ZHD518;

[0032] Figure 4 It is the thermal stability graph of the recombinant enzyme S1v1-GS-ZHD518. Detailed Description of the Invention

[0033] To better understand the present invention, the content of the present invention will be further clarified below in conjunction with embodiments. However, the content of the present invention is not limited to the following embodiments. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in the technical field.

[0034] The method for measuring the enzyme activity of zearalenone-degrading enzyme: The total reaction system is 210 μl, including 5 μl of 2.5 mg / ml ZEN, 5 μl of the dialyzed enzyme solution (0.2 mg / ml), and 190 μl of PBS buffer; after reacting at 40 °C for 5 min, 50 μl of 1 M HCl and 300 μl of methanol are added to terminate the reaction. Take the supernatant, pass it through a 0.22 nm organic filter membrane, and then take 20 μl of the sample to detect on a high performance liquid chromatograph (Agilent 1260, fluorescence detector). The sample is eluted with 60% acetonitrile at a flow rate of 0.8 ml / min, and detected under the conditions of an excitation wavelength of 274 nm, an emission wavelength of 440 nm, and a column temperature of 30 °C through an Agilent ZORBAX SB-C18 chromatographic column (4.6 mm × 150 mm, 5 μm). Calculate the amount of the residual substrate ZEN according to the peak area under the HPLC elution curve. The enzyme activity is defined as the amount of enzyme required to reduce 1 μmol of the substrate per minute of reaction as 1 U.

[0035] Example 1 In vitro expression of the recombinant enzyme S1v1-GS-ZHD518

[0036] 1. Construction of the expression vector

[0037] The self-assembling amphiphilic short peptide (S1v1, AEAEAHAHAEAEAHAH) was fused to the N-terminus of the polypeptide sequence encoded by the ZEN-degrading gene ZHD518 (GenBank accession number, XM_013418296.1) via the (GSSS)3 linker peptide, and a 6×His tag was retained at the C-terminus. The target fragment (shown in SEQ ID NO.2) was cloned into the expression vector pET22b(+) using the restriction sites of NdeI and HindⅢ to obtain the recombinant plasmid (i.e., pET22b-S1v1-GS-ZHD518).

[0038] 2. Expression and purification

[0039] The recombinant plasmid pET22b-S1v1-GS-ZHD518 was transformed into competent E. coli BL21(DE3) cells by heat shock for expression. Subsequently, the recombinant E. coli was cultured in 50 ml of LB medium containing Amp (100 mg / ml) at 37°C and 220 rpm for scale-up culture until the OD 600 reached 0.6 - 0.8. Then, IPTG with a final concentration of 0.2 mM was added to the medium, and the culture was incubated at 16°C and 150 rpm for 24 h to induce the production of the S1v1-GS-ZHD518 enzyme. After induction, the culture broth was centrifuged at 9000 rpm for 10 min to collect the cells. The cells were then resuspended in lysis buffer (50 mM PBS, 10 mM imidazole, pH 7.5) and lysed using an ultrasonic cell disruptor in an ice-water bath (ultrasonic for 2 s, pause for 3 s) until a clear liquid, i.e., the crude enzyme solution, was obtained. The lysed liquid was centrifuged (9000 rpm, 10 min) to remove the lysis precipitate, and the lysate supernatant was obtained by filtering through a 0.22 μm filter membrane. The obtained lysate supernatant was passed through a Ni-NTA column, and the unbound miscellaneous proteins were removed using the wash buffer (50 mM PBS, 20 mM imidazole, pH 7.5) and the recombinant protein S1v1-GS-ZHD518 was purified using the elution buffer (50 mM PBS, 100 mM imidazole, pH 7.5). Finally, imidazole was removed by dialysis. The eluate was collected in aliquots and subjected to SDS-PAGE electrophoresis. The SDS-PAGE electrophoresis results of the recombinant protein are as Figure 1 shown, indicating that the recombinant protein S1v1-GS-ZHD518 with high purity was successfully purified.

[0040] Example 2 Detection of the expression effect of the recombinant enzyme

[0041] The expression level and enzyme activity of the recombinant enzyme constructed in Example 1 were detected.

[0042] Table 1 shows the changes in the expression level, total enzyme activity, and specific activity of purified S1v1-GS-ZHD518. Compared with the crude enzyme, the specific activity of the purified enzyme increased by 4.4-fold, reaching 6.03 U / mg. After the original ZHD518 was purified by nickel column, the total protein content was 5.5 mg, and the specific activity was 0.65 U / mg. Overall, the enzyme activity of S1v1-GS-ZHD518 was 9.27 times higher than that of the wild type. After S1v1 was fused with ZHD518, the expression level, total enzyme activity, and specific activity were all significantly improved.

[0043] Table 1 Purification results of S1v1-GS-ZHD518

[0044]

[0045] Example 3 Determination of the optimal pH of recombinant enzyme S1v1-GS-ZHD518

[0046] The recombinant enzyme S1v1-GS-ZHD518 was reacted in reaction buffers with pH 3.0, 4.6, 6.6 (citrate-sodium citrate buffer), pH 6.0, 7.0, 8.0 (phosphate buffer), pH 7.5, 8.0, 8.5, 9.0 (Tris-HCl buffer), pH 8.6, 9.6, 10.6 (glycine-sodium hydroxide buffer) for 10 min to determine its optimal reaction pH value. The enzyme activity under the condition of pH 8.0 (Tris-Hcl buffer) was taken as 100%. All experiments were carried out in triplicate, and the experimental results were averaged.

[0047] The results are as Figure 2 shown. S1v1-GS-ZHD518 had relatively high enzyme activity at pH 7 - 9.6, which was 5.03 - 6.03 U / mg; the enzyme activity was optimal at pH 8. Under acidic conditions, the enzyme activity decreased significantly. When the pH value was 6, the remaining activity was only 60.57% of the maximum value, and when the pH value dropped to 3, the remaining activity further decreased to less than 30%.

[0048] Example 4 Determination of the optimal temperature of recombinant enzyme S1v1-GS-ZHD518

[0049] The reaction buffer (PBS, pH 7.5) containing the substrate was incubated at 25, 30, 35, 40, 45, 50, 55, 60 °C for 2 min, and then the recombinant protein S1v1-GS-ZHD518 was added to react for 10 min at different temperature conditions to determine its optimal reaction temperature.

[0050] As Figure 3As shown, at 35 - 45 °C, the enzyme activity of the fusion enzyme is 5.82 - 5.92 U / mg. The protein activity is the highest at 40 °C. After this temperature, the enzyme activity gradually decreases. The activity of the protein is only 71.66% of the highest activity at 55 °C and 34.78% of the highest activity at 60 °C. The original enzyme shows relatively high enzyme activity (specific enzyme activity is 0.39 - 0.65 U / mg) within a narrow temperature range of 30 - 40 °C and drops sharply by about 80% at 45 °C. In contrast, the recombinant enzyme fused with the functional peptide S1v1 maintains relatively high enzyme activity within a wider temperature range of 40 - 55 °C.

[0051] Example 5 Determination of the Thermal Stability of Recombinant Enzyme S1v1 - GS - ZHD518

[0052] The recombinant protein S1v1 - GS - ZHD518 was heat - treated at 30, 35, 40, 45, 50, 55, and 60 °C for 5, 10, 15, and 30 min. After the treatment, the protein solution was immediately cooled in 4 °C, and then its thermal stability was determined.

[0053] The results are as Figure 4 shown. The enzyme activity of S1v1 - GS - ZHD518 remains basically unchanged under the conditions of 40 - 45 °C. On the contrary, when the original enzyme ZHD518 was heated at 45 °C for 10 min, the activity decreased to 25%. In contrast, the thermal stability of S1v1 - GS - ZHD518 was increased by 37.08 times when heated at 45 °C for 10 min. After the recombinant enzyme was incubated at 50 °C for 15 minutes, the enzyme activity remained basically unchanged, and after 30 minutes, the enzyme activity remained at 72.362%. In addition, even after being exposed at 55 °C for 15 minutes, the enzyme still retained 47.548% of its activity. However, at 60 °C, the remaining activity of the enzyme is generally below 30%, and after incubating for 30 minutes, the activity of the enzyme is basically lost.

[0054] Example 6 Enzyme Kinetics Determination of Recombinant Enzyme S1v1 - GS - ZHD518

[0055] To determine the catalytic reaction rate and substrate - binding ability of the modified enzyme, the kinetic parameters (Vmax, Km, Kcat) of S1v1 - GS - ZHD518 were calculated using the Michaelis - Menten model. The determination was carried out by using different concentrations of ZEN substrate (0 - 350 μM).

[0056] Table 2 Enzyme Kinetics of S1v1 - GS - ZHD518

[0057]

[0058]

[0059] Example 7 Determination of the Degradation Effect of Recombinase S1v1-GS-ZHD518 on ZEN in Corn Bran, Corn Germ Meal and Corn Gluten Meal

[0060] Actual samples such as corn germ meal, corn bran, and corn gluten meal were obtained from a small corn by-product processing workshop. The solid samples were ground and sieved through a 40-mesh sieve. For enzymatic ZEN degradation, 200 μg of the purified fusion enzyme S1v1-GS-ZHD518 prepared according to the method of Example 1 was added to a reaction containing 5 g of corn by-product sample and 30 mL of PBS (pH 7.5) in a 50 mL centrifuge tube (the inactivated enzyme was used as the control group). The reaction was carried out at 45 °C and 180 rpm for 60 minutes. The samples after the reaction were dried overnight in a freeze dryer, and then 3 mL of extraction solution (acetonitrile: water: acetic acid = 70:29:1) was added to the solution in 3 portions, and ultrasonic extraction was carried out for 20 minutes in sequence. 1 mL aliquots of the supernatant were transferred to 1.5 mL centrifuge tubes, dried for 1 h in a freeze dryer, redissolved in 0.5 mL of 60% acetonitrile in water, and then the impurities were removed with a 0.22 μm organic filter membrane. Finally, ZEN was detected by HPLC.

[0061] Table 3 Degradation Effect of Recombinase S1v1-GS-ZHD518 on ZEN in Corn Bran, Corn Germ Meal and Corn Gluten Meal

[0062]

[0063] Comparative Example 1:

[0064] A biological decontaminant mainly composed of Clostridium butyricum, Bacillus subtilis and Enterococcus faecalis was used as a control. The biological decontaminant was purchased from Anhui Best Biotechnology Co., Ltd., product number: Wan Si Tian (2023) H01008. According to the method of Example 1, ZEN in corn gluten meal (the same product as in Example 7, initial ZEN content 5.296 μg / g) was removed, and the reaction was carried out at 45 °C and 180 rpm for 15, 30, and 60 minutes respectively.

[0065] Comparative Example 2:

[0066] A domestic detoxifying agent, Meili Jie, was used as a control. Meili Jie was purchased from Henan Yiwan Zhongyuan Biotechnology Co., Ltd., product number: Yu Si Tian Zi (2017) 089001. According to the method of Example 1, ZEN in corn gluten meal (the same product as in Example 7, initial ZEN content 5.296 μg / g) was removed, and the reaction was carried out at 45 °C and 180 rpm for 15, 30, and 60 minutes respectively.

[0067] Table 4 ZEN Degradation Rates of Corn Gluten Meal under Different Reaction Conditions

[0068]

[0069] Although the present invention has been disclosed above with the preferred embodiments, it is not intended to limit the present invention. Any person skilled in this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be defined by the claims.

Claims

1. A fusion enzyme of zearalenone degrading enzyme, characterized in that, The amino acid sequence is as shown in SEQ ID NO.

1.

2. The gene encoding the fusion enzyme according to claim 1.

3. The gene according to claim 2, wherein The nucleotide sequence is as shown in SEQ ID NO.

2.

4. A recombinant Escherichia coli, characterized in that, Using pET22b as the expression vector to express the fusion enzyme with the amino acid sequence as shown in SEQ ID NO.

1.

5. A method for preparing the fusion enzyme according to claim 1, characterized in that, Culturing the recombinant Escherichia coli according to claim 4 and collecting the fusion enzyme in the cell culture medium.

6. The method according to claim 5, characterized in that, Culturing the recombinant Escherichia coli according to claim 4 in LB medium for a period of time, inducing with IPTG, and collecting the fusion enzyme in the bacterial cells.

7. The application of the fusion enzyme according to claim 1 in the degradation of zearalenone.

8. The application according to claim 7, characterized in that, The application is to degrade zearalenone in corn by-products; the corn by-products include corn hulls, corn germ meal or corn gluten meal.

9. The application of the fusion enzyme according to claim 1, or the recombinant Escherichia coli according to claim 4 in the food field or the feed processing field.

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