Preparation method and application of Anammox bacteria sulfide quinone oxidoreductase

Abstract

The invention discloses a preparation method and application of Anammox bacteria sulfide quinone oxidoreductase, and belongs to the technical field of bioengineering. The enzyme is derived from anaerobic ammonia oxidation Anammox bacteria, the nucleotide sequence of the enzyme is as shown in SEQ ID NO 1, and the amino acid sequence of the enzyme is as shown in SEQ ID NO 2. The preparation method comprises the following steps: 1) designing and synthesizing a primer for amplifying a sulfide quinone oxidoreductase gene; 2) carrying out PCR amplification and constructing a recombinant plasmid; and 3) expressing and purifying the sulfide quinone oxidoreductase. Anmmox bacteria containing the enzyme can be widely applied to the fields of sewage treatment, environmental restoration, biosensing and the like. The bacterium not only can efficiently convert ammonia nitrogen and nitrite in wastewater into nitrogen, but also can synergistically reduce the concentration of sulfide in the wastewater, reduce the pollution to the environment, improve the wastewater treatment efficiency and reduce the operation cost. The enzyme still has high denitrification activity under oxygen-containing and salt-containing conditions.

Description

Technical Field

[0001] The invention belongs to the technical field of bioengineering, and in particular relates to a preparation method and application of Anammox bacteria sulfide quinone oxidoreductase obtained by genetic engineering means. Background Art

[0002] Nitrogen and sulfur pollution are two major challenges facing current environmental governance. Nitrogen pollution mainly comes from processes such as agriculture, urban life and industrial production, including the application of agricultural chemicals, livestock and poultry breeding waste and industrial wastewater discharge. Excessive nitrogen can lead to eutrophication of water bodies, trigger red tides and algal blooms, destroy the balance of aquatic ecosystems, and even cause "dead zones" in water bodies. At the same time, sulfur pollution is mainly related to the treatment of wastewater, waste gas and waste from industrial production, mineral processing and chemical processes. These activities produce a large amount of sulfide, which spreads into the environment through groundwater and soil pollution. High concentrations of sulfide not only produce a foul odor, but also irritate the respiratory system and eyes, and long-term exposure may endanger human health. Therefore, how to effectively remove nitrogen and sulfur pollutants in sewage has become an important task in current environmental governance. Traditional denitrification and desulfurization technologies, such as biological filtration, adsorption, incineration, chemical and biological deodorants, etc., can solve these pollution problems to a certain extent, but there are limitations such as secondary pollution, high energy consumption and high cost. Therefore, there is an urgent need to develop green, economical and efficient sewage harmless treatment technologies to provide better solutions to nitrogen and sulfur pollution.

[0003] Sulfide:quinone oxidoreductase (SQR) is an enzyme that converts sulfides (including H2S, HS - , S 2- ) is oxidized to zero-valent sulfur (S 0), and transfers electrons to quinone through the cofactor FAD, reducing it to a peripheral membrane protein called hydroquinone. The active center of SQR contains the FAD cofactor, which plays a key catalytic role in this process. SQR is an ancient riboflavin protein belonging to the disulfide oxidoreductase family. It is widely present in various organisms except plants, including photoautotrophic and chemoautotrophic bacteria, archaea, and animal cells. In 1994, Ariel et al. isolated and purified SQR from the cell membrane of the cyanobacterium Oscillatoria limnetica and found that it was a 57kDa polypeptide that could be cloned and expressed in E. coli. In the following 20 years, 13 SQR proteins from different sources were isolated, expressed and purified, providing an important basis for exploring its structure, function and regulatory mechanism. It is worth noting that SQR can catalyze the oxidation reaction of sulfide in the extracellular environment without entering the cell. This feature makes SQR have broad application prospects in application fields such as sewage treatment. Through in-depth research on the structure, function and regulatory mechanism of SQR, it is expected to develop new biotechnology based on SQR and provide innovative solutions for wastewater desulfurization.

[0004] Anaerobic ammonium oxidation (Anammox) has attracted widespread attention as an emerging biological denitrification technology. The Anammox process uses specialized anaerobic ammonium oxidation bacteria to directly convert ammonia nitrogen and nitrite in wastewater into harmless nitrogen gas without the need for exogenous organic carbon and oxygen supply. This unique biochemical mechanism gives Anammox many environmental advantages: first, it can efficiently remove nitrogen pollutants in wastewater with low energy consumption, greatly reducing the operating cost of wastewater treatment; second, the Anammox process does not produce greenhouse gases and plays a positive role in addressing climate change; in addition, Anammox also avoids the secondary pollution problem caused by traditional nitrification-denitrification processes, reflecting the characteristics of being more green and environmentally friendly. It is precisely based on these significant advantages that Anammox technology has been widely used in the treatment of high-concentration nitrogen-containing industrial wastewater and agricultural wastewater. However, in the treatment of complex water bodies such as urban sewage, Anammox technology still faces some challenges. First, how to quickly enrich highly active Anammox bacteria is the key to achieving stable and efficient denitrification; second, the existing Anammox enrichments have very limited tolerance to harsh environmental conditions, and how to optimize the reaction conditions to improve their adaptability is also a major problem; in addition, most Anammox bacteria can only function in anaerobic, low-sulfur freshwater environments, so screening and cultivating Anammox strains that can adapt to complex environments is also an urgent problem to be solved. Summary of the invention

[0005] The purpose of the present invention is to provide an sqr gene derived from Anammox bacteria and an optimized expression vector of the sqr gene in view of the above-mentioned deficiencies in the prior art. A recombinant vector capable of efficiently expressing the sqr gene is constructed by using the highly pure and adaptable Anammox bacteria enriched from mangrove sediments.

[0006] Another object of the present invention is to provide a method for determining the enzymatic characteristics of SQR and verifying its physiological function of promoting nitrogen metabolism of Anammox bacteria.

[0007] In order to achieve the above-mentioned invention object, the present invention provides the following technical solutions:

[0008] A sulfide:quinone oxidoreductase (SQR) from Anammox bacteria, the nucleotide sequence of which is shown in SEQ ID NO 1, and the amino acid sequence of which is shown in SEQ ID NO 2;

[0009] The optimum temperature of the Anammox bacteria sulfide quinone oxidoreductase is 37° C., the optimum pH is 8.0, and the optimum salinity is 0.5% NaCl.

[0010] The preparation method of the Anammox bacteria sulfide quinone oxidoreductase comprises the following steps:

[0011] 1) Construction of a heterologous expression system for efficient expression of SQR protein:

[0012] E. coli was selected as the heterologous expression host to take advantage of its high expression efficiency and simple operation; the pET-28a vector that can be used for screening and induced expression was used to facilitate the identification and expression optimization of the recombinant plasmid; the sqr gene from Anammox bacteria was cloned into the multiple cloning site of pET-28a to construct a recombinant expression vector; this recombinant vector was transferred into the E. coli BL21 host strain to construct a transgenic strain that can efficiently express the SQR protein.

[0013] 2) Establish an efficient purification method for SQR protein:

[0014] 6×His tags were added to the N-terminus and C-terminus of the SQR protein, respectively, and the high affinity with the nickel affinity chromatography resin was used to achieve rapid purification of the SQR protein. This method can obtain highly pure and active recombinant SQR protein, providing a guarantee for subsequent enzymatic property analysis and physiological function verification.

[0015] 3) Conduct enzymatic characterization analysis of SQR protein:

[0016] Detect the redox activity of SQR protein on sulfide under alkaline conditions to explore its optimal enzyme activity; evaluate the kinetic parameters of SQR protein to provide a basis for further optimizing its physiological function in Anammox metabolism;

[0017] 4) Verify the physiological function of SQR protein in Anammox bacteria:

[0018] The purified recombinant SQR protein was added to the Anammox bacterial culture system to observe its effect on the denitrification efficiency of Anammox bacteria; it was proved that SQR protein can significantly promote the nitrogen metabolism of Anammox bacteria, providing key support for the development of efficient Anammox technology.

[0019] In step 1), the following aspects are taken into consideration when heterologously expressing the SQR protein: 1. Use highly efficient and universal strains as much as possible to ensure sufficient and stable protein expression; 2. Select a vector plasmid that can be screened and induced for expression, which is convenient for identification of recombinant plasmids and subsequent large-scale expression of recombinant proteins; 3. For subsequent protein purification, select a suitable protein purification tag. A protein tag refers to a polypeptide or protein that is fused and expressed with the target protein using DNA in vitro recombination technology to facilitate the expression, detection, tracing and purification of the target protein. The protein tag can be a Flag tag, a His tag, an MBP tag, an HA tag, a GST tag, a SUMO tag, etc.

[0020] A method for amplifying a sqr gene comprises the following steps:

[0021] 1) Design and synthesize primers for amplifying sqr, the sequences of which are as follows:

[0022] Upstream primer SQR0009F:

[0023] 5'-CGCGGATCCATGGGGATCAGACATCAT-3'

[0024] Downstream primer SQR0009R:

[0025] 5'-CCCAAGCTTTTAAGGTTTTCCTTTCAA-3'

[0026] 2) PCR amplification and construction of recombinant plasmid:

[0027] Amplification was performed using primers SQR0009F and SQR0009R with genomic DNA of high-purity Anammox bacteria (SRS16676642) as a template; the PCR amplified product was connected to the pET-28a expression vector digested with restriction endonucleases BamH I and Hind III, and the connection product was transformed into Escherichia.coli; the PCR product was digested with restriction endonucleases BamH I and HindIII and then connected to the expression vector pET-28a digested with the same restriction endonucleases; the connection product was transformed into E.coli DH5α, and the recombinant plasmid containing the sqr gene was screened, and nucleic acid electrophoresis and sequencing were performed for verification;

[0028] The conditions of the PCR amplification were as follows: 95°C pre-denaturation for 5 min, 95°C for 30 s, 52°C for 30 s, 72°C for 90 s, 72°C for 10 min, and 35 cycles.

[0029] 3) Expression and purification of SQR protein

[0030] The recombinant plasmid containing the sqr gene was transformed into E.coli BL21, and the SQR protein expression was induced by low-concentration IPTG at low temperature, and then the protein was purified to obtain the SQR protein; the purification was performed by Ni-NTA agarose affinity chromatography.

[0031] The invention discloses an expression vector of sulfide quinone oxidoreductase of Anammox bacteria. The expression vector is constructed by inserting a sqr gene from Anammox bacteria into a multiple cloning site of an expression vector pET-28a.

[0032] A SQR transgenic E. coli strain is constructed by transforming the above SQR expression vector into BL21.

[0033] The invention provides a purification method for heterologously expressed SQR protein. 6×His tags are added to both ends of the SQR protein, and the affinity between the tags and a nickel column is utilized to purify the protein.

[0034] The present invention provides a method for determining the enzymatic characteristics of SQR protein under alkaline conditions, by detecting S 2- The consumption is measured.

[0035] The present invention also provides a method for detecting the function of the Anammox bacteria sulfide quinone oxidoreductase protein at a physiological level, and proves the promoting effect of the Anammox bacteria sulfide quinone oxidoreductase protein on the denitrification of Anammox bacteria.

[0036] An Anammox bacterium containing the sqr gene can be widely used in sewage treatment, environmental remediation and biosensing.

[0037] The present invention is the first to heterologously express the SQR protein encoding gene in Anammox bacteria and prove the effect of SQR protein on the denitrification of Anammox bacteria. As an important component of Anammox metabolism, the heterologous and efficient expression of SQR provides key experimental materials for in-depth revelation of the biological characteristics of Anammox bacteria. Through a comprehensive study of the enzymatic characteristics and physiological functions of the purified SQR protein, the important role of SQR in the nitrogen metabolism of Anammox bacteria is clarified, laying a solid foundation for the further development of efficient and novel Anammox technology.

[0038] The efficient SQR protein expression and purification technology system constructed by the present invention can be widely used in the functional research of Anammox-related enzyme proteins. Based on the green denitrification mechanism of Anammox bacteria, the technology of the present invention is expected to promote the innovative application of Anammox technology in the fields of sewage treatment and environmental remediation, and help achieve sustainable development goals. In addition, high-purity SQR proteins can also be used for basic research in the fields of biosensing and electron transfer, providing new technical support for the development of related industrial chains.

[0039] The present invention utilizes highly pure and adaptable Anammox bacteria enriched from mangrove sediments to construct a recombinant vector capable of efficiently expressing the sqr gene. Through purification and enzymatic characteristic studies, the physiological function of SQR in Anammox bacteria is further verified. Key experimental materials and technical support are provided for further development of a new Anammox technology with both denitrification and desulfurization capabilities. The purity of the enriched Anammox bacteria is above 90%, and it carries a unique sqr gene (rarely found in existing Anammox bacteria), and still has a denitrification efficiency of more than 60% under high salinity and oxygen conditions. Therefore, the present invention is expected to lay the foundation for the development of a new Anammox technology with both denitrification and desulfurization capabilities. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 Signal peptide prediction for SQR protein;

[0041] Figure 2 Prediction of transmembrane region of SQR protein;

[0042] Figure 3 To predict the tertiary structure of SQR protein;

[0043] Figure 4 Alignment of SQR protein sequence with other homologous sequences;

[0044] Figure 5 This is the result of PCR amplification of the sqr gene of Anammox bacteria;

[0045] Figure 6Schematic diagram of the pET-28a-SQR recombination process;

[0046] Figure 7 PCR identification of BL21 bacterial solution of pET-28a-SQR recombinant plasmid. 1: Negative control; 2-7: PCR amplification of recombinant plasmid pET-28a-SQR;

[0047] Figure 8 The SDS-PAGE electrophoresis diagram of the SQR protein Ni-NTA column purification product; 1: control group with an empty plasmid containing no sqr gene; 2: cell lysis solution without IPTG induction; 3: cell lysis solution without centrifugation after induction; 4: precipitate after ultrasonic crushing and centrifugation; 5: supernatant after ultrasonic crushing and centrifugation; 6-7: washing buffer for eluting impurities for the first and fourth times; 8-9: elution buffer for eluting SQR protein twice;

[0048] Fig. 9 Standard curve of bovine serum albumin content drawn for Bradford method;

[0049] Fig.10 CuSO4 method for determination of S 2- Standard curve of content;

[0050] Fig.11 It is a line graph of the relative activity of SQR at different temperatures;

[0051] Fig.12 It is the relative activity line graph of SQR at different pH values;

[0052] Fig.13 It is the relative activity line graph of SQR at different NaCl concentrations;

[0053] Fig.14 is the nonlinear enzyme activity kinetic curve of SQR under the optimal conditions;

[0054] Fig.15 The effect of SQR physiological function on the denitrification efficiency of Anammox bacteria. DETAILED DESCRIPTION

[0055] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the following embodiments will further illustrate the present invention in conjunction with the accompanying drawings. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0056] Example 1 Preparation of SQR:

[0057] 1. Experimental materials:

[0058] Strain: Anammox high-purity enrichment was obtained by enrichment for 3 years in a top-down flow reactor built by the applicant's laboratory. Plasmid pET-28a vector, E. coli DH5α and BL21 competent cells were purchased from Takara.

[0059] The main enzymes and kits used in the experiment: plasmid mini-extraction kit HiPure Plasmid Micro Kit C, agarose gel DNA recovery kit HiPure Gel Pure DNA Mini Kit were purchased from Magen; DNA extraction kit DNeasy PowerSoil Pro Kit was purchased from QIAGEN; Bradford protein quantification kit Bradford Protein Assay Kit was purchased from Biotechnology; Ni-NTA resin, protein purification buffer Binding / Washing buffer For Ni-NTA and Elution buffer For Ni-NTA were purchased from Biotech; BamH I, Hind III restriction endonucleases, 10×QuickCut Green Buffer, T4 DNA Ligase, 10×T4 DNA Ligase Buffer were purchased from TaKaRa; other common medicines and chemical reagents were of domestic analytical grade.

[0060] 2. SQR protein structure prediction:

[0061] For the assembled genome of Anammox bacteria (SAMN33053686) on NCBI GenBank, the nucleotide sequence of the sqr gene SEQ ID NO 1 was 1206 bp and the protein sequence SEQ ID NO 2 was 403 bp after annotation by software such as Prokka.

[0062] In order to improve the efficiency of protein expression and purification and subsequent functional studies, the SQR protein structure was first predicted, and the signal peptide and transmembrane domain of the protein were predicted using SignalP 5.0 and TMHMM 2.0. Figure 1 and Figure 2 As shown in Figure 2, it indicates that SQR has no signal peptide and transmembrane region, that is, it is a non-secretory protein. Then the SQR protein structure was predicted, and the results are as follows Figure 3 As shown: SQR contains two absolutely conserved cysteines to form a catalytic disulfide redox center and relies on surface flavin adenine nucleotide (FAD) to catalyze sulfide.

[0063] 3. Cloning of the sqr gene

[0064] According to the nucleotide sequence of the sqr gene, primers were designed using Primer 6.0 and Oligo 7.0 software to amplify the sqr sequence from Anammox bacteria. The primer sequences are as follows:

[0065] SQR0009F: 5'-CGC GGATCC ATGGGGATCAGACATCAT-3' (the underlined part is the BamHI restriction site)

[0066] SQR0009R: 5'-CCC AAGCTT AGGTTTTCCTTTCAA-3' (the underlined part is the HindⅢ restriction enzyme recognition site)

[0067] According to the kit instructions, the genomic fragment of Anammox bacteria was extracted as a template, and PCR amplification was performed using the specific primers designed above. Amplification reaction conditions: 95℃ pretreatment for 5min, 95℃ for 30s, 52℃ for 30s, 72℃ for 90s, 35 cycles, and 72℃ extension for 10min to obtain the sqr gene from Anammox bacteria. PCR reaction system (50μL): Forward primer 2μL; Reverse primer 2μL; Template DNA 1μL; 2×Hieff PCR Master MIX 25μL; ddH2O 21μL. The amplified product was subjected to 1% agarose gel electrophoresis to detect that the size of the sqr gene was consistent with the theoretical value, which was approximately 1200bp ( Figure 4 ).

[0068] 4. Construction of pET-28a-SQR and identification of recombinant bacteria

[0069] Use restriction endonucleases BamH I and Hind III to double-digest the sqr gene fragment and pET-28a, then use a recombinant cloning kit to connect the target gene to the vector, mix the ligation product with the recipient bacteria DH5α competent cell, slowly invert and flick the tube wall to mix, and immediately place on ice for 30 minutes. Heat shock in a 42°C water bath for 45 seconds, then place on ice for 2 to 3 minutes, and add 900 μL of LB culture medium without antibiotics. Incubate at 37°C in a shaker at 200rpm for 2 to 3 hours, centrifuge to remove 90% of the supernatant, then resuspend the cells, and evenly spread the resuspended bacterial solution on the flat solid culture medium with a sterile coating rod. Incubate inverted in a 37°C incubator for 12 to 16 hours. The monoclonal colonies grown on the overnight culture plate may be recombinant bacteria that have been successfully transformed. By electrophoresis ( Figure 6 ) and sequencing to screen the recombinant plasmids.

[0070] 5. Construction of BL21 expression strain for recombinant protein

[0071] The recombinant plasmid in 4 above was re-transformed into BL21 competent cells to complete the construction of recombinant bacteria and obtain a strain containing SQR.

[0072] 6. Expression and purification of SQR protein

[0073] 1) SQR protein induction: IPTG induction is added at low temperature in the present invention to make the BL21 strain express more SQR protein. The specific induction operation is as follows: before IPTG induction expression, the BL21 bacteria containing the pET-28a-SQR recombinant plasmid are inoculated into LB liquid culture medium with a kanamycin concentration of 100 μg / mL, and cultured at 37°C until the absorbance value OD600 of the bacterial liquid is 0.6-0.8, and then ITPG is added to make the final concentration in the culture medium 0.5mM, and induced at 16°C for 16h.

[0074] 2) SQR protein acquisition:

[0075] After induction, the supernatant of the bacterial solution and the cells were centrifuged, the supernatant was discarded, and the cells were washed twice with PBS. According to the weight of the cells, 10 mL of lysis buffer (Binding buffer used for purification) was added to resuspend the cells at a ratio of 1 g, and the protease inhibitor PMSF was added to control the final concentration to 1 mM. The bacterial sample was then ultrasonically disrupted, the power was set to 25%, and the lysis was performed for 15 minutes until the sample became clear and transparent from turbidity. Centrifuge at 10,000 rpm for 5 minutes, discard the precipitate, and retain the supernatant, which contains active SQR recombinant protein.

[0076] 3) SQR protein purification

[0077] Gravity column filling and pretreatment: Add an appropriate amount of Ni-NTA resin to the separation column, let the preservation solution flow out naturally, and complete the gravity column filling. Add two times the column volume of Binding / Wash Buffer, mix the buffer and resin thoroughly to balance the nickel column, let it stand for 1 to 2 minutes, and let the buffer flow out naturally.

[0078] Sample loading and protein binding: Mix the protein extract with Binding / Wash Buffer at a ratio of 1:1, so that the total volume of the mixture is equivalent to twice the volume of the resin. Add the protein mixture to the resin, mix the mixture and the resin, and let the protein and resin bind for 5-10 minutes, inverting and mixing several times during the period. Allow the bound mixture to flow out naturally under the action of gravity, and control the flow rate at 0.5mL / min-1mL / min; if there is excess protein mixture, it can be passed through the column again for binding.

[0079] Elution of impurity proteins: Use Binding / Wash Buffer twice the volume of the resin to elute the non-specifically bound impurity proteins, and allow the eluate to flow out naturally. Save the eluate separately to analyze the elution of impurity proteins; repeat the elution step 2 to 3 times.

[0080] Elution of SQR protein: Use Elution Buffer twice the volume of the resin to elute the Ni-bound histidine-tagged protein on the resin, and control the flow rate at 0.5 mL / min-1 mL / min. To obtain a higher concentration of protein, you can elute again. Repeat the steps twice to obtain more SQR recombinant protein. Perform SDS-PAGE electrophoresis on the eluate from each step of the purification process to detect protein expression.

[0081] Figure 1 The signal peptide prediction of SQR protein shows the result of signal peptide prediction of SQR protein using SignalP 5.0 software. It can be clearly seen in the figure that there is no obvious signal peptide region in the sequence of SQR protein, which indicates that SQR protein is not a secretory protein, but may play its function in the cell. Figure 2 Prediction of the transmembrane region of the SQR protein; This figure uses TMHMM 2.0 software to predict the transmembrane domain of the SQR protein. The results show that there is no transmembrane region in the SQR protein sequence, which further supports the hypothesis that the SQR protein is an intracellular protein. Figure 3 The tertiary structure prediction of SQR protein is shown in the figure. Figure 3 As can be seen in the figure, the SQR protein contains two absolutely conserved cysteines that may form catalytic disulfide redox centers. In addition, the SQR protein also relies on surface flavin adenine nucleotide (FAD) to catalyze sulfide. This structural feature is crucial for understanding the catalytic mechanism of the SQR protein. Figure 4 is the alignment of the SQR protein sequence with other homologous sequences; Figure 5 It is the result of PCR amplification of sqr gene of Anammox bacteria; Figure 5 The results of PCR amplification of the sqr gene of Anammox bacteria using specific primers are shown. A 1200 bp amplified band can be clearly seen in the figure, which is consistent with the expected size of the sqr gene. This shows that the present invention successfully amplified the sqr gene of Anammox bacteria.

[0082] Figure 6 The figure is a schematic diagram of the pET-28a-SQR recombination process, including the amplification, double enzyme digestion, ligation, transformation and screening of the sqr gene. It can be seen that the present invention successfully constructs the pET-28a-SQR recombinant plasmid. Figure 7PCR identification of BL21 bacterial culture of pET-28a-SQR recombinant plasmid; Figure 7 The results of identifying the pET-28a-SQR recombinant plasmid using the PCR method are shown. Figure 7 Figure 1 is a negative control, and Figures 2-7 are the results of PCR amplification of the recombinant plasmid pET-28a-SQR. It can be seen that samples 2-7 all showed amplification bands consistent with expectations, indicating that the present invention successfully constructed the pET-28a-SQR recombinant plasmid and transformed it into BL21 competent cells.

[0083] Figure 8 This is the SDS-PAGE electrophoresis of the SQR protein Ni-NTA column purification product. Figure 8 The SDS-PAGE electrophoresis results of SQR protein purification using Ni-NTA column are shown. Figure 8 1 is the control group into which the empty plasmid without sqr gene was introduced, 2 is the cell lysis solution without IPTG induction, 3 is the cell lysis solution without centrifugation after induction, 4 is the precipitate after ultrasonic crushing and centrifugation, 5 is the supernatant after ultrasonic crushing and centrifugation, 6-7 are the results of the first and fourth elution of impurities in the washing buffer, and 8-9 are the results of the elution buffer for two elutions of the SQR protein. By comparing the results of each group, the purification process of the SQR protein and its presence in the elution solution can be clearly seen.

[0084] During the purification process, in order to verify the purification effect and determine the protein concentration, a Bradford protein quantification kit (purchased from Beyotime) was used for protein quantification. Fig. 9 This is the standard curve of bovine serum albumin content drawn by Bradford method, which is used to determine the concentration of SQR protein in subsequent experiments. By measuring the absorbance value of unknown samples and comparing it with the standard curve, the concentration of purified SQR protein can be calculated.

[0085] Example 2 Enzymatic properties of SQR

[0086] 1. Method for determining the activity of SQR catalytic sulfide

[0087] The present invention selects Na2S as the substrate, performs the in vitro catalytic reaction of SQR under anaerobic conditions, and measures the S 2- The activity of the enzyme was calculated by measuring the consumption of 2- The experimental principle of the content is as follows: Under alkaline conditions, the S 2- Basically, it exists in the solution in a free state. It reacts with copper sulfate solution to form copper sulfide, which is brown-black and can be measured at 480nm. 2- Standard concentration curve of content ( Fig.10 ), the absorbance value of the sample to be tested can be used to determine the S before and after the reaction. 2- consumption.

[0088] 2. Effect of temperature on SQR activity

[0089] At pH 8.0, the S 2- The enzyme activity was calculated, and the highest activity was taken as 100% relative enzyme activity. The relative enzyme activity under the remaining conditions was calculated to determine the effect of temperature on SQR enzyme activity. The results showed that SQR had the highest activity at 37°C, indicating that 37°C was the optimal enzyme reaction temperature ( Fig.11 ).

[0090] 3. Effect of pH on SQR activity

[0091] At 37°C, the S 2- The consumption of CuSO4 can be used to determine the effect of pH on enzyme activity. 2- The concentration can only be carried out under alkaline conditions, and S under neutral and acidic conditions 2- Will generate HS - and H2S, which made it impossible to accurately measure, so the experiment was carried out under alkaline conditions, and the pH values ​​of each group were set to 8.0, 9.0, 10, and 11. The experimental results showed that the optimal reaction pH value under alkaline conditions was 8.0, and SQR could still maintain a high activity under pH 9.0-10.0 ( Fig.12 ).

[0092] 4. Effect of salinity on SQR activity

[0093] At 37°C and pH 8.0, reaction systems with NaCl concentrations of 0%, 0.1%, 0.5%, 1.0%, 1.5%, and 3.0% were prepared to measure S under different salinity conditions. 2- The results showed that the optimal reaction salinity was 0.5% NaCl concentration, and the activity of the enzyme could still be maintained at 40% at a salinity of 3.0%. Fig.13 ).

[0094] 5. Determination of the mechanical parameters of SQR enzyme activity

[0095] After determining the optimal reaction conditions through the above experiments, the enzyme kinetic parameters were measured at 37°C, pH 8.0, and salinity 0.5%. 2-The substrate concentration was 50, 100, 150, 200, and 250 μM. After a period of reaction, S 2- The consumption of the enzyme was calculated and the enzyme reaction rate was calculated. With the substrate concentration as the horizontal axis and the reaction rate as the vertical axis, the nonlinear kinetic curve was fitted in combination with the Michaelis-Menten equation ( Fig.14 ), the enzyme activity of SQR was 0.89 U / mg, and the Michaelis constant K m The maximum enzymatic reaction rate V max It was 6.078±0.288μmol / L·min, indicating that the purified SQR had a good substrate affinity.

[0096] 6. Verification of the physiological function of SQR in Anammox bacteria

[0097] This study found that the highly purified Anammox bacteria obtained have a high tolerance to oxygen and still have a denitrification efficiency of 60% at an oxygen concentration of 12.5% ​​(v / v). In order to verify whether SQR can help Anammox bacteria resist oxidative stress, this study designed two groups of experiments. One group was normal culture of Anammox bacteria under different oxygen concentrations, and the second group was cultured on the basis of the first group with the addition of purified SQR protein (final concentration of 0.1 mg / mL). After 24 hours of culture in both groups, 1 mL of solution was taken out from the culture system, and the supernatant was centrifuged to detect the concentrations of ammonia nitrogen and nitrite nitrogen. Using a spectrophotometer, according to the absorbance of ammonia nitrogen and nitrite nitrogen at specific wavelengths (420nm, 540nm), the concentrations of ammonia nitrogen and nitrite nitrogen in the sample were calculated by measuring the standard solution to obtain a standard curve. Fig.15 As shown in the figure, Anammox bacteria can still denitrify at an oxygen concentration of 10% (v / v, about 151 μmol / L). Compared with the normal group, the removal of total nitrogen was significantly improved after adding SQR protein at five different oxygen concentrations. This shows that SQR can help Anammox bacteria resist oxidative stress and promote its better growth to denitrify efficiently.

[0098] The invention uses the high-purity genomic DNA of Anammox bacteria from mangroves as a template, designs and synthesizes amplification primers, amplifies the sqr gene, clones the gene into the pET-28a expression vector, clones it in E.coli DH5α, and then transfers it into E.coli BL21 for induced expression, and uses Ni-NTA agarose affinity chromatography to purify to obtain high-purity recombinant SQR. The enzyme has a wide range of alkalinity and salinity, can maintain more than 40% activity under conditions of strong alkalinity and high salinity, and has good stress resistance. The physiological function verifies that the protein can promote Anammox bacteria to still perform Anammox reaction under aerobic conditions. Therefore, Anammox bacteria containing SQR can play a role in an environment with high salinity, oxygen and high sulfur concentration, which is not only expected to solve the problem of sewage treatment in coastal cities, but also provide key microbial resources and technical support for the development of a new Anammox technology with both denitrification and desulfurization capabilities. Since the enzyme has high denitrification activity under oxygenated and saline conditions, it also has application potential in desalination plants, marine aquaculture farms, and other salinity wastewater treatment facilities.

[0099] The above embodiments are only preferred embodiments of the present invention and cannot be considered to limit the scope of the present invention. All equivalent changes and improvements made within the scope of the present invention should still fall within the scope of the present invention.

Claims

1. Anammox sulfide quinone oxidoreductase, characterized in that The nucleotide sequence is shown in SEQ ID NO 1, and the amino acid sequence is shown in SEQ ID NO 2.

2. The method for preparing the Anammox sulfide quinone oxidoreductase according to claim 1, characterized in that The following steps are involved: a) constructing a heterologous expression system for efficiently expressing SQR protein, selecting E. coli as a heterologous expression host, using a pET-28a vector that can be screened and induced to express, cloning the sqr gene from Anammox bacteria into the multiple cloning site of pET-28a, constructing a recombinant expression vector, and transferring the recombinant vector into an E. coli BL21 host strain to construct a transgenic strain that can efficiently express SQR protein; b) Establishing an efficient purification method for SQR protein, adding 6×His tags to the N-terminus and C-terminus of the SQR protein, respectively, and using nickel affinity chromatography resin to achieve rapid purification of the SQR protein; c) Conduct enzymatic characterization of SQR protein, detect the redox activity of SQR protein on sulfide under alkaline conditions, and evaluate its kinetic parameters; d) To verify the physiological function of the SQR protein in Anammox bacteria, the purified recombinant SQR protein was added to the Anammox bacteria culture system to observe its effect on the denitrification efficiency of Anammox bacteria.

3. The method for preparing the Anammox sulfide quinone oxidoreductase according to claim 2, characterized in that In step a), when heterologous expression of SQR protein is performed, a highly efficient and universal bacterial species is selected, a vector plasmid that can be screened and induced for expression is selected, and a suitable protein purification tag is selected, wherein the protein purification tag includes a Flag tag, a His tag, an MBP tag, an HA tag, a GST tag and a SUMO tag.

4. A method for amplifying the sqr gene, characterized in that The following steps are involved: 1) Design and synthesize primers for amplifying sqr, the sequences of which are as follows: Upstream primer SQR0009F: 5'-CGCGGATCCATGGGGATCAGACATCAT-3' Downstream primer SQR0009R: 5'-CCCAAGCTTTTAAGGTTTTCCTTTCAA-3' 2) PCR amplification and construction of recombinant plasmid: Use primers SQR0009F and SQR0009R, use genomic DNA of highly purified Anammox bacteria (SRS16676642) as a template, and perform PCR amplification using the primers described in step 1); The PCR amplified product was connected to the pET-28a expression vector digested with restriction endonucleases BamH I and Hind III, and the connection product was transformed into Escherichia.coli. The PCR product was digested with restriction endonucleases BamH I and Hind III and then connected to the expression vector pET-28a digested with the same restriction endonucleases. The connection product was transformed into E.coli DH5α, and the recombinant plasmid containing the sqr gene was screened, and nucleic acid electrophoresis and sequencing were performed for verification; 3) Expression and purification of SQR protein The recombinant plasmid containing the sqr gene was transformed into E.coli BL21, and the SQR protein expression was induced by low-concentration IPTG at low temperature, and then the protein was purified to obtain the SQR protein; the purification was performed by Ni-NTA agarose affinity chromatography.

5. A method for amplifying the sqr gene as claimed in claim 4, characterized in that The conditions for the PCR amplification were: pre-denaturation at 95°C for 5 min, 95°C for 30 s, 52°C for 30 s, 72°C for 90 s, 72°C for 10 min, and 35 cycles.

6. An expression vector of Anammox sulfide quinone oxidoreductase, characterized in that the expression vector is constructed by inserting the sqr gene from Anammox into the multiple cloning site of the expression vector pET-28a.

7. Use of Anammox bacteria containing the Anammox bacteria sulfide quinone oxidoreductase according to claim 1 in sewage treatment.

8. Use of Anammox bacteria containing the Anammox bacteria sulfide quinone oxidoreductase according to claim 1 in environmental remediation.

9. Use of Anammox bacteria containing the Anammox bacteria sulfide quinone oxidoreductase according to claim 1 in biosensing.

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