Sulfamethoxazole-degrading enzyme derived from wheat and use thereof

Abstract

The application discloses a sulfamethoxazole degrading enzyme derived from wheat and an application thereof, and belongs to the field of plant pollutant degradation. A key gene POD of wheat for degrading SMX is identified, and the coding sequence of the POD gene is shown as SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3 or SEQ ID NO. 4. The POD gene is also recombinantly expressed in E. coli, and the POD enzyme is used for SMX degradation in an in-vitro environment. The application provides a scientific basis for evaluating the toxic effect of SMX on crops, and also provides a theoretical reference for the application of plants for degrading SMX in the environment.

Description

TECHNICAL FIELD

[0001] The present application relates to sulfamethoxazole-degrading enzyme derived from wheat and its application, and belongs to the field of plant pollutant degradation. BACKGROUND

[0002] Sulfamethoxazole (SMX) is a broad-spectrum sulfonamide antibiotic. Currently, SMX is widely used to treat various bacterial infections. However, SMX cannot be completely metabolized by organisms, and it can enter wastewater, agricultural runoff and water environment with excreta, thereby polluting the environment. Current studies have shown that the concentration of SMX in surface water is 0.47 mg L -1 , and the concentration of SMX in groundwater is as high as 0.48 mg L -1 . With the growth of population and the intensification of global climate change, in order to meet the needs of citizens, industry and agriculture, various water sources containing SMX are used for agricultural irrigation and domestic water, thus posing potential risks of polluting the environment and food. In 2017, the International Agency for Research on Cancer (IARC) published a preliminary list of carcinogens, and SMX was listed in the list of class 3 carcinogens.

[0003] Plants have the function of accumulating and degrading antibiotics, and therefore, plants have been applied to the removal of various environmental pollutants, such as organic pollutants, heavy metals and antibiotics. Wetland plants such as water hyacinth, cattail and reed can accumulate and degrade sulfonamide antibiotics. However, studies have shown that while plants degrade sulfonamide antibiotics, antibiotics also have an undeniable impact on the growth and development of plants. For example, Kong, Li et al. showed that SMX can inhibit the growth and photosynthesis of alfalfa, Arabidopsis and cucumber. So far, the phytotoxicity of SMX has been mainly studied in animals and model plants. SMX existing in irrigation water and soil also poses a safety hazard to food crops. Food crops are the main food for the world's population and are closely related to human health, but there is no clear SMX safety concentration standard for crop growth, and there is no research on the accumulation and degradation mechanism of SMX in food crops. Therefore, the safety risk of SMX in food crops needs to be evaluated. SUMMARY

[0004] The purpose of the present application is to clarify sulfamethoxazole-degrading enzyme derived from wheat and its application, to provide a scientific basis for evaluating the toxic effects of SMX on crops, and to provide a theoretical reference for the application of plants to degrade environmental SMX.

[0005] The present application provides the application of POD enzyme in degrading sulfamethoxazole.

[0006] In one embodiment, the application is to degrade sulfamethoxazole in a non-animal environment.

[0007] In an embodiment, the application is to express the POD enzyme in plants to degrade sulfamethoxazole pollution.

[0008] In an embodiment, the gene encoding the POD enzyme is as shown in any one of SEQ ID NO. 1-4.

[0009] In an embodiment, the plant includes but is not limited to wheat.

[0010] The application also provides the use of the POD enzyme in resisting sulfamethoxazole pollution and promoting plant growth.

[0011] In an embodiment, the promotion of plant growth includes but is not limited to promoting seed germination, increasing root length, increasing shoot length, and increasing plant fresh weight.

[0012] In an embodiment, the plant includes but is not limited to crops.

[0013] In an embodiment, the plant is wheat.

[0014] In an embodiment, the concentration of sulfamethoxazole is ≥0.1 mg / L.

[0015] In an embodiment, the concentration of sulfamethoxazole is 0.1-5 mg / L.

[0016] Advantages:

[0017] (1) The application uses different concentrations of SMX to treat wheat seeds to evaluate the toxic effects of SMX on wheat growth. The degradation ability of wheat to SMX is determined, the key gene POD of wheat degrading SMX is identified, and the effects of SMX on wheat growth and the key gene of wheat degrading SMX are elucidated. The application determines that SMX has a certain effect on wheat seedling germination, growth and material accumulation, and it is also determined that wheat has the ability to degrade SMX, and the function verification of the key gene POD of wheat degrading SMX is completed.

[0018] (2) The application also recombinantly expresses the POD gene in E. coli, and uses the POD enzyme for SMX degradation in vitro.

[0019] The application provides a scientific basis for evaluating the toxic effects of SMX on crops, and also provides a theoretical reference for the application of plants degrading environmental SMX. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1To improve the effect of sulfamethoxazole concentration on the average germination rate, average sprout length, root length, sprout dry weight and root dry weight of wheat seedlings. Among them, A: the effect of sulfamethoxazole concentration on the average germination rate of wheat seedlings; B: the effect of sulfamethoxazole concentration on the average sprout length and root length of wheat seedlings; C: the effect of sulfamethoxazole concentration on the average sprout dry weight and root dry weight of wheat seedlings.

[0021] Figure 2 To analyze the SMX degradation effect of wheat sterile seedlings and wild seedlings. Among them, A: SMX content of sterile seedlings and treatment solution treated with SMX; B: SMX content of wild seedlings and treatment solution treated with SMX; C: SMX degradation rate of sterile seedlings and wild seedlings treated with SMX.

[0022] Figure 3 To analyze the key enzyme of wheat degrading SMX. Among them, A: SOD activity in sterile wheat seedlings under different SMX concentrations; B: PPO activity in sterile wheat seedlings under different SMX concentrations; C: POD activity in sterile wheat seedlings under different SMX concentrations; D: APX activity in sterile wheat seedlings under different SMX concentrations; E: CAT activity in sterile wheat seedlings under different SMX concentrations; F: GR activity in sterile wheat seedlings under different SMX concentrations.

[0023] Figure 4 To analyze the SMX degradation effect of POD in sterile wheat seedlings and wild seedlings.

[0024] Figure 5 To analyze the SMX degradation effect of the protein obtained by overexpressing E. coli POD. DETAILED DESCRIPTION

[0025] The specific embodiments of the present application are further described in detail by the description of the examples, to help the skilled in the art to have a more complete, accurate and in-depth understanding of the inventive concept and technical solutions of the present application.

[0026] (1) Preparation of plant material and sulfamethoxazole

[0027] The wheat (Triticum aestivum) variety Luyuan 502 with stable genetic traits was provided by Shandong Academy of Agricultural Sciences. The variety is widely planted in central China. The surface of sterile seed was disinfected with 5% sodium hypochlorite (NaClO) solution for 15 minutes, and then washed with water to remove excess NaClO. The wild seed was washed with water. Then, the seeds were placed in a culture dish with two wet filter papers for germination for 12 hours. The sulfamethoxazole used in the specific embodiments of the present application (purchased from Shanghai Shengong Biological Engineering Co., Ltd., China) was diluted to 0.1, 0.5, 1 and 5 mg L -1The concentration of the enzyme solution was determined and stored in a brown bottle for later use.

[0028] (2) Determination of the degradation ability of enzyme solution to sulfamethoxazole

[0029] Extraction of crude enzyme solution from wheat seedlings: The experimental material was ground and 0.2 g of plant sample was accurately weighed and ground in liquid nitrogen. It was then placed in a 5 mL centrifuge tube and 2 mL of pure methanol was added for extraction. The sample was then placed in an ultrasonic cleaner (40 Hz) and ultrasonicated for 1 h. After centrifugation at 10000 r for 5 min, the supernatant was collected.

[0030] 1 mL of the crude enzyme solution was passed through anhydrous sodium sulfate column and then filtered through a 0.22 μm microporous filter before being measured by liquid chromatography (HPLC, Agilent 1260 Infinity II, Santa Clara, California, USA). The liquid phase conditions were as follows: the stationary phase was Agilent C 18 (4.6 mm * 250 mm, 5 μm), and the mobile phase was 0.1% formic acid solution mixed with organic phase acetonitrile at a ratio of 70:30. After degassing, it was used in the high-performance liquid chromatograph. The chromatographic conditions were as follows: flow rate 1.0 ml / min, column temperature 30°C, detector UV detector, detection wavelength 270 nm, and injection volume 10 μL. The external standard method was used, and the content of sulfamethoxazole was calculated by comparing the peak area.

[0031] (3) Determination of the degradation enzyme activity in wheat

[0032] Superoxide dismutase (SOD) activity was determined by nitro blue tetrazolium (NBT) spectrophotometry (reference: Effects of diethyl aminoethyl hexanoate on seed germination characteristics of white clover under chromium stress, published in 2021). The reaction mixture included 50 mM PBS (pH 7.6), 13 mM methionine, 75 μM NBT, 0.1 mM EDTA, and an appropriate amount of enzyme extract. After irradiation at 25°C for 30 min, the absorbance was recorded at 560 nm. The uncolored reaction medium was used as a dark control. One unit of enzyme activity refers to the amount of enzyme that inhibits the reduction rate of NBT by 50% at a wavelength of 560 nm. The enzyme activity is represented by U (min g FW) -1 .

[0033] Peroxidase (POD activity was determined by oxidation of guaiacol in the presence of H2O2 (refer to the paper published in 2021, Effects of diethyl aminoethyl hexanoate on seed germination characteristics of white clover under chromium stress). The enzyme extract was mixed with 3 cm PBS (50 mM, pH 7.0) containing 20 mM guaiacol. The mixture was incubated at 25°C for 5 minutes, and then 6 μM H2O2 was added to start the reaction. The change in absorbance was recorded at 470 nm wavelength at 20-second intervals for 2 minutes, and then the POD activity was calculated with an absorbance coefficient of 26.6 mM-1cm-1. The POD activity of the enzyme extract was expressed in U (min g FW) -1 .

[0034] Catalase (CAT) activity was determined using ultraviolet absorbance (refer to the paper published in 2021, Effects of diethyl aminoethyl hexanoate on seed germination characteristics of white clover under chromium stress). CAT activity was detected by the decrease in absorbance at 240 nm wavelength. The reaction mixture with a total volume of 2 mL contained 25 mM sodium phosphate buffer (pH 7.0) and 10 mM H2O2. 100 μL of enzyme extract was added at the start of the reaction, and the initial disappearance rate of H2O2 was measured at 240 nm wavelength (E = 39.4 mM-1cm-1) after 30 seconds to determine the CAT activity of the enzyme extract, which was expressed in U (min g FW) -1 .

[0035] Polyphenol oxidase (PPO) activity was determined by visible spectrophotometry. In a test tube containing 4 ml of 0.2 M sodium phosphate dibasic-0.1 M citric acid buffer with pH 5.8, 0.05 ml of 0.05 M catechol solution was added, and after preheating in a 30°C constant temperature water bath, 0.05 ml of enzyme solution was added, and the reaction was allowed to proceed for 5 minutes, and the absorbance value was read at 410 nm on a spectrophotometer. Under the above conditions, the A410 reading was used, and an increase of 0.01 OD value per minute was defined as one unit of enzyme activity, U (min g FW) -1 .

[0036] Ascorbate peroxidase (APX) activity was determined with reference to the paper published in 2021, Effects of diethylaminoethyl hexanoate on seed germination characteristics of white clover under chromium stress. The phenomenon of APX reducing the amount of ascorbic acid in the presence of H2O2 was used to accurately measure the activity of the enzyme. The 3 ml reaction mixture contained 50 mmol / L sodium phosphate buffer (pH 7.0), 0.1 mmol / L EDTA-Na2, 0.3 mmol / L AsA, 0.06 mmol / L H2O2 and 0.1 ml enzyme solution. After adding H2O2 to start the reaction, the change in A290 was measured at 20°C within 10-30 seconds, and the amount of AsA reduced per unit time and the enzyme activity were calculated, with the unit being U (min g FW) -1 .

[0037] Glutathione peroxidase (GSH-Px) activity was determined with reference to the paper published in 2021, Effects of diethylaminoethyl hexanoate on seed germination characteristics of white clover under chromium stress. The total volume of the reaction mixture was 3 mL and contained 50 mmol / L sodium phosphate buffer (pH 7.0), 0.1 mmol / L EDTA-Na2, 1 mmol / L glutathione disulfide (GSSG), 0.2 mmol / L NADPH and 0.1 ml enzyme extract. The reaction was started by adding 0.1 ml enzyme extract, and immediately the change in absorbance (A340) at 340 nm was measured using a spectrophotometer within 10-30 seconds to determine the GSH-Px activity of the enzyme extract, with the unit being U (min g FW) -1 .

[0038] Example 1 Effect of sulfamethoxazole on the growth of wheat

[0039] Wheat seeds were randomly divided into three groups of 100 seeds each and transplanted into 12 cm diameter glass bottles containing two sheets of moistened filter paper. Each seed was exposed to 25 mL of a sulfamethoxazole solution at three different concentrations (0.1 g / L, 0.5 g / L, and 1 g / L). The negative control group was exposed to distilled water for the same time period. Treatments were repeated three times. All experimental groups were cultured in a plant growth chamber at 25°C under dark / light cycling (8 / 16 hours). The treatment solution was changed daily to maintain a constant concentration. Seed germination rate was defined as the number of germinated seeds on day 7 after the start of cultivation. After two weeks of growth, root length, shoot length, fresh weight, and dry weight were measured in 30 seedlings from each group. Each experiment was repeated three times.

[0040] To analyze the effects of SMX on wheat seedling germination, growth, and nutrient accumulation, root length, shoot length, fresh weight, and dry weight of wheat seedlings were measured. The results are as follows: Figure 1 As shown, the germination rate of wheat first decreased and then increased with increasing SMX concentration. SMX significantly affected the root and shoot lengths of wheat at concentrations of 0.1 mg / L. -1 Under treatment, the levels increased compared to the control, but the difference was not significant, although the concentration was ≥0.5 mg / L. -1 The root length and shoot length of the wheat that grew after this period were significantly lower than those of the control group (p<0.01). Figure 1 B). After measuring the amount of dry matter accumulation, the results showed that with the increase of SMX concentration, the dry weight of shoots and roots also showed a trend of first increasing and then decreasing, with the lowest concentration at 0.1 mg / L. -1 Under treatment, the dry weight of shoots and roots increased compared to the control, but the difference was not significant, although the concentration was ≥0.5 mg / L. -1 The dry weight of the buds and roots was significantly lower than that of the control group (p<0.01). Figure 1 C). Similar to most antibiotics and organic pollutants, SMX is also toxic to wheat. The concentration of SMX is 0.1 mg / L. -1 At that time, the germination rate, growth, and nutrient accumulation of wheat seedlings were all increased to some extent compared with the control, suggesting that wheat has a certain degradation effect on SMX, and the degradation products of the benzene ring have a certain promoting effect on wheat germination and growth. (SMX concentration ≥ 0.5 mg / L) -1Subsequently, wheat germination rate, growth, and dry matter accumulation significantly decreased (p<0.01), which is speculated to be due to the following reasons: First, high concentrations of SMX damaged the seed coat function, allowing large amounts of SMX to enter the seed interior, inhibiting seed germination and the synthesis of nutrients during later growth; second, SMX entered the plant, leading to damage and loss of function of root tip meristems and chloroplasts; third, SMX can induce an increase in ROS in wheat roots, causing plasma membrane damage and inhibiting the activity of various oxidases and invertases. This will lead to conformational changes in various biomolecules, ultimately affecting wheat growth, nutrient and water absorption, thereby inhibiting wheat growth and dry matter accumulation.

[0041] Example 2: Study on the SMX degradation ability of wheat

[0042] To clarify the degradation capacity of wheat for SMX, the SMX content and degradation rate in wheat treated with SMX for 12 hours according to the method in Example 1 and in the treatment solution were determined. Figure 2 The SMX concentration was 0.1 mg / L. -1 At that time, SMX was not detected in sterile and wild-grown wheat seedlings or in the treatment solution. Figure 2 A~ Figure 2 B) indicates that wheat achieved a 100% degradation rate of this concentration of SMX. Figure 2 C). This may also be an important reason why wheat growth was not inhibited at this concentration. SMX concentration ≥ 0.5 mg / L -1 Subsequently, SMX was detected in both wheat cells and the treatment solution, and the concentration increased with increasing treatment concentration. Figure 2 A, Figure 2 B). Extraction and analysis of SMX in wheat revealed a very low SMX content, below 0.02% of the initial total SMX, indicating that, like wild seedlings, sterile wheat seedlings primarily remove SMX through degradation and do not accumulate it within the plant. The SMX content in sterile and wild wheat seedlings at 0.1 mg / L... -1 The SMX degradation rate was 100% at 0.5 mg / L. -1 Under SMX treatment, the degradation rates of SMX by wild seedlings and sterile seedlings were 93% and 87%, respectively. Although the degradation rate of SMX by wild seedlings was higher than that by sterile seedlings, the difference was not significant. This indicates that the bacteria have a certain degradation effect on SMX, but it has no significant effect on the SMX degradation rate of wheat.

[0043] Example 3: Analysis of key enzymes in wheat that degrade SMX

[0044] The degradation of pollutants by plants mainly relies on some antioxidant enzyme systems that have the ability to degrade exogenous substances. Therefore, the main antioxidant enzymes (SOD, POD, CAT, APX, PPO and GSH-Px) in the crude enzyme solution of aseptic wheat seedlings treated with SMX according to the method of Example 1 were determined and analyzed. Figure 3

[0045] The results show that the SOD activity decreases with the increase of SMX treatment concentration Figure 3 A), the PPO activity does not change significantly with the increase of SMX treatment concentration Figure 3 B), indicating that SOD and PPO are not sensitive to SMX and are not the key enzymes for degrading SMX. The activities of POD, CAT, APX and GSH-Px increase first and then decrease with the increase of SMX treatment concentration Figure 3 C ~ Figure 3 F). Under the treatment of 0.1 mg L -1 SMX, the activity of POD increases by 50%, the activity of APX increases by 5.56%, the activity of CAT increases by 11.25%, and the activity of GSH-Px increases by 28.82%. When the SMX concentration is ≥0.5 mg L -1 , the activities of SOD, PPO, POD and CAT decrease significantly, and the activities of APX and GSH-Px increase. Combined with the degradation rate of SMX by wheat and the size of enzyme activity, it is speculated that POD is the key degradation enzyme in the process of wheat degrading SMX.

[0046] Example 4 Verification of the key enzyme of wheat degrading SMX

[0047] In order to further clarify whether POD is the key enzyme of wheat degrading SMX, the POD enzyme protein of wheat with the same mass (0.2 g) was extracted for in vitro degradation verification.

[0048] Under the treatment of 0.1 mg / L SMX, the degradation rates of POD of wild seedlings and aseptic seedlings to SMX are 90% and 88% Figure 4 , respectively, which is not significantly different from the SMX degradation rate of wheat seedlings; when the SMX concentration is ≥0.5 mg L -1 , the SMX degradation rate of POD decreases, but the overall degradation rate is still higher than 60%, indicating that POD is the key enzyme of wheat degrading SMX.

[0049] ​According to the literature and the conserved sequence of the wheat POD gene published in Genbank, the wheat POD (POD-1, POD-2, POD-3, POD-4) gene sequence (nucleotide sequences are shown in SEQ ID NO. 1-4) is obtained. The plant-derived POD is expressed by using the E. coli expression system. The specific steps are as follows: the POD-1, POD-2, POD-3, POD-4 gene sequences (shown in SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4) are cloned into the pET-28a(+) vector to obtain the recombinant plasmids pET-28a(+)-POD-1, pET-28a(+)-POD-2, pET-28a(+)-POD-3, pET-28a(+)-POD-4.

[0050] The wheat POD belongs to the heme-containing type III plant peroxidase, which catalyzes the oxidation of various aromatic compounds with H2O2 as the electron acceptor. The expression ability of the wheat POD in E. coli is weak, and the main reason is that the content of heme synthesized by E. coli is insufficient, and the enzyme structure and enzyme activity of the POD require a large amount of heme. The synthesis of heme by E. coli adopts the C5 pathway, and glutamyl tRNA is used as the precursor, and finally heme is formed through 7 intermediate products. The first step of this synthesis pathway, i.e., the reaction of synthesizing 5-ALA from glutamyl tRNA, is the rate-limiting step, which is catalyzed by the glutamyl tRNA reductase (GluTR, Hem A) and the glutamate semialdehyde mutase (GSAM, Hem L) encoded by the hemA and hemL genes. In order to improve the functional expression of the POD in E. coli, the hemA and hemL genes (nucleotide sequences are shown in SEQ ID NO. 5, SEQ ID NO. 6) are cloned into the pACYC vector to obtain the recombinant plasmid pACYC-A-L, and the recombinant plasmid pACYC-A-L containing the hemA and hemL genes is co-transformed into the E. coli BL21 (DE3) strain with the pET-28a(+)-POD-1, pET-28a(+)-POD-2, pET-28a(+)-POD-3, pET-28a(+)-POD-4 containing the POD gene, so as to enhance the functional expression of the POD in the prokaryotic system.

[0051] The constructed recombinant E. coli is cultured in LB medium at 37℃ for 14-16h, and the culture solution of the recombinant cells is collected by centrifugation at a speed of 5000r / min for 5min, and then resuspended with PBS, and the resuspension is broken in a high-pressure homogenizer at 6℃, 600Pa for 15min. After breaking, the supernatant is separated by centrifugation at 10 000r / min for 15min to obtain the enzyme protein solution.

[0052] The collected enzyme protein was added to a 20 mL system containing 5 mg / L SMX at a final concentration of 10 mg / L fresh weight enzyme concentration of wheat, and reacted at 30°C, 4000XL light for 4 h. The results showed that the expressed POD-1, POD-2, POD-3, and POD-4 proteins had degradation ability to 5 mg / L SMX, and the SMX degradation rates of the POD-1, POD-2, POD-3, and POD-4 enzyme liquids were 35%, 54%, 21%, and 33%, respectively. Figure 5

[0053] Although the present application has been disclosed with reference to the preferred embodiments as above, it is not intended to limit the present application, and any person skilled in the art can make various modifications and modifications without departing from the spirit and scope of the present application, and therefore the protection scope of the present application should be defined by the claims.​

Claims

1. Use of a POD enzyme in the degradation of sulfamethoxazole, characterized in that, The coding gene of the POD enzyme is shown as SEQ ID NO. 1-4.

2. Use according to claim 1, characterized in that, The POD enzyme is expressed in plants.

3. Use according to claim 1 or 2, characterized in that, The plants include but are not limited to wheat.

4. Use of POD enzymes for combating sulfonamidomethoxazole contamination and for promoting plant growth, characterized in that, The coding gene of the POD enzyme is shown as SEQ ID NO. 1-4.

5. Use according to claim 4, characterized in that, The promotion of plant growth includes but is not limited to: promoting seed germination, increasing root length, increasing shoot length, increasing plant fresh weight.

6. Use according to claim 5, characterized in that, The plants include but are not limited to crops.

7. Use according to claim 5, characterized in that, The plants are wheat.

8. Use according to any one of claims 4 to 7, characterized in that, The concentration of sulfamethoxazole is ≥0.1 mg / L.

9. Use according to any one of claims 4 to 7, characterized in that, The concentration of sulfamethoxazole is 0.1-1 mg / L.

10. A method of degrading sulfamethoxazole, characterized by, The POD enzyme is contacted with sulfamethoxazole in the environment; the coding gene of the POD enzyme is shown as SEQ ID NO. 1-4.

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