Preparation method of halophilic archaea nitrate reductase and application thereof

Abstract

The application belongs to the field of genetic engineering, and particularly relates to a preparation method and application of a novel halophilic archaea nitrate reductase. Hor The constructed recombinant plasmid pTA04-nas is transformed into a prokaryotic host, and after culture expansion, bacterial bodies are collected by centrifugation; cells are broken and lysed by ultrasonic, and after centrifugation, supernatant is collected for subsequent purification; after purification by nickel column affinity chromatography and concentration by ultrafiltration, Nas Hor , has excellent enzymatic properties, especially wide adaptability to salinity, temperature and pH, and has better salinity, temperature and pH stability, and good tolerance to metal ions and organic solvents. Hor The nucleotide sequence is shown as SEQ ID NO. 2, and the amino acid sequence is shown as SEQ ID NO. 1; Nas Hor The application can be applied to the degradation of nitrate in pickled vegetables and sewage, and provides a novel enzyme preparation for the degradation of nitrate in high-temperature, high-salt and alkaline environments, and has a wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering, specifically relating to a novel method for preparing nitrate reductase from halophilic archaea and its application. Background Technology

[0002] Nitrates are naturally present in plant-based foods, and reports have shown that various vegetables, especially leafy vegetables, are contaminated with nitrates to varying degrees. Nitrates themselves are harmless or have low hazard, but they can be reduced to harmful nitrites during fermentation or in the human body, causing "secondary harm" from nitrites. Utilizing nitrate reductases to degrade nitrates offers advantages such as specificity and high efficiency. Currently reported nitrate reductases are mainly derived from plants and bacteria, limiting their application under extreme conditions. Halophilic archaea are microorganisms that typically inhabit high-salt environments such as salt pans, natural salt lakes, and pickled products. Due to the specificity of their habitat, enzymes derived from halophilic archaea usually exhibit characteristics such as salt and alkali tolerance, heat resistance, and resistance to organic solvents. Halophilic archaea provide excellent strain resources for the search of euryhaline nitrate reductases.

[0003] Nitrate reductase catalyzes the reduction of nitrate to nitrite. Currently published halophilic archaea nitrate reductases can be classified according to their physiological functions into assimilatory nitrate reductases (Nas), dissimilatory nitrate reductases (Nap), and respiratory nitrate reductases (Nar). Nas, located in the cytoplasm, is easy to collect and purify, making it a focus of attention in applied fields. Nas can be further divided into ferrugin- or xanthoredoxin-dependent Nas and NADH-dependent Nas. Ferroredoxin-dependent Nas is mainly a 75–85 kDa monomer, while NADH-dependent Nas is mainly a heterodimer.

[0004] Assimilatory nitrate reductases from halophilic archaea have only been reported in strains Haloferax alexandrinus and Haloferax mediterranei, but the purification steps used are cumbersome and yield low results. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of existing technologies and provide a simple method for preparing nitrate reductase (Nas) from a halophilic archaea (Halorarius litoreus) and its application in pickling vegetables and wastewater treatment. The halophilic archaea used in this invention is designated as Halorius litoreus BND22. T The halophilic archaeal nitrate reductase involved in this invention exhibits good stability under high temperature, high salt, and alkaline conditions. The purification and performance testing of Nas provide a basis for the application of halophilic archaeal nitrate reductase, and provide a novel enzyme preparation for degrading nitrates in pickled vegetables and wastewater.

[0006] The halophilic archaea of ​​this invention was purchased from the China General Microbiological Culture Collection Center, accession number CGMCC1.18780; this invention is based on the halophilic archaea (Halorarius litoreus BND22). T A novel halophilic archaea nitrate reductase gene, named nas, was obtained from the genome of a halophilic archaea. Hor Based on NAS Hor The novel halophilic archaea nitrate reductase encoded was named Nas Hor Its nucleotide sequence is shown in SEQ ID NO.2, and its amino acid sequence is shown in SEQ ID NO.1.

[0007] This invention relates to a method for preparing nitrate reductase from halophilic archaea, specifically comprising the following steps:

[0008] (1) Construction of recombinant plasmid pTA04-nas Hor ;

[0009] Based on the halophilic archaea (Halorarius litoreus BND22) T Functional alignment of the whole genome sequence of nas, a halophilic archaea nitrate reductase gene, was obtained. Hor Then, the target gene fragment nas was amplified using PCR technology. Hor Then, using molecular cloning technology, NAS Hor The recombinant plasmid pTA04-nas was constructed by ligating it into the pTA04 vector. Hor ;

[0010] (2) Recombinant plasmid pTA04-nas Hor Transformation of prokaryotic hosts and expression;

[0011] The recombinant plasmid pTA04-nas constructed in step (1) Hor The recombinant cells were transformed into prokaryotic hosts, inoculated into Hv-YPC liquid medium, cultured until the growth stabilized, and then centrifuged to collect the cells for storage.

[0012] (3) Purification of nitrate reductase from halophilic archaea;

[0013] The bacterial cells collected in step (2) were resuspended in cell lysis buffer. After resuspending, the cells were sonicated to disrupt the cell structure. The supernatant was collected after centrifugation and purified using nickel affinity chromatography. Subsequently, the enzyme solution was concentrated by ultrafiltration to obtain high-purity halophilic archaea nitrate reductase, denoted as Nas. Hor .

[0014] Preferably, the halophilic archaea mentioned in step (1) is derived from the China General Microbiological Culture Collection Center, with the culture collection number CGMCC 1.18780.

[0015] Preferably, the prokaryotic host in step (2) is based on the strain Haloferax volcanii, and a mutant strain is constructed to finally obtain the uracil-thymidine auxotrophic mutant strain, denoted as Haloferax volcanii H1424; its construction method is referenced from: Allers T, Ngo HP, Mevarech M, et al. Development of Additional Selectable Markers for the Halophilic Archaeon Haloferax volcanii Based on the leuB and trpA Genes[J]. Applied and Environmental Microbiology, 2004, 70(2): 943-953. The prokaryotic host is Haloferax volcanii, which was purchased from the Japan Microbial Culture Collection Center, and the culture collection number is JCM 8879.

[0016] Preferably, the Hv-YPC culture medium in step (2) consists of the following components (per liter): 5.0 g yeast extract, 1.0 g soybean peptone, 1.0 g acid-hydrolyzed casein, 4.2 g KCl, 33.0 g MgSO4·7H2O, 30.0 g MgCl2·6H2O, 144.0 g NaCl, 12.0 mL 1M Tris-HCl (pH 8.0), and 0.33 g CaCl2 (pre-dissolved in distilled water and added after dissolution). The above components are brought to a final volume of 1 L with distilled water, the pH is adjusted to 7.5, and the medium is sterilized at 115 °C for 30 min.

[0017] Preferably, the culture conditions in step (2) are 37°C, 200 rpm, and culture for 3 to 4 days; the preservation is low-temperature preservation at -20°C.

[0018] Preferably, the cell lysis buffer in step (3) consists of 2M NaCl, 20mM Tris-HCl, and pH 8.0.

[0019] Preferably, the steps of nickel column affinity chromatography in step (3) are as follows: drain the 20% ethanol stored in the nickel column, wash the column with 10 column volumes of ddH2O, and then equilibrate the column with 10 column volumes of cell lysis buffer; flow the supernatant collected by centrifugation into the nickel column and let it stand for 10 min; elute the impurities with 10 column volumes of buffer I; and elute the target protein with 5 column volumes of buffer II.

[0020] Preferably, the buffer solution I consists of 40 mM imidazole, 2 M NaCl, 20 mM Tris-HCl, and pH 8.0.

[0021] Preferably, the buffer II consists of 100 mM imidazole, 2 M NaCl, 20 mM Tris-HCl, and pH 8.0.

[0022] Preferably, the ultrafiltration in step (3) is concentrated using an ultrafiltration centrifuge tube with a molecular weight cutoff of 10 kDa.

[0023] The halophilic archaea nitrate reductase prepared by this invention can provide a novel enzyme preparation for the degradation of nitrate in high-salt, high-temperature, and alkaline environments, and can be specifically applied to the degradation of nitrate in pickled vegetables and sewage.

[0024] Performance testing:

[0025] Enzymatic characterization of nitrate reductase in halophilic archaea;

[0026] This invention Nas Hor The enzyme properties were characterized, specifically including optimal temperature and temperature stability; optimal pH and pH stability; optimal NaCl concentration and NaCl concentration stability; organic solvent tolerance; metal ion tolerance and determination of enzyme kinetic parameters.

[0027] Using NaNO3 as a substrate, Nas Hor The optimal reaction conditions are 70℃, 2.0M NaCl, and pH 9.0. It exhibits good thermal stability at both 70℃ and 90℃; excellent tolerance to both low-salt and high-salt environments; and good pH stability at both pH 8.0 and pH 10.0. This enzyme is effective against Fe... 3+ and K + It exhibits good tolerance; ethanol, glycerol, and isopropanol have varying degrees of promoting effect on the enzyme activity. When NaNO3 is used as a substrate, Nas... Hor Maximum reaction rate V max The kinetic constant K is 30.94 μg / min / mg. m It is 1.64 mM.

[0028] Advantages and technical effects of the present invention:

[0029] (1) This invention discovers a novel halophilic archaea nitrate reductase gene, expresses a novel halophilic archaea nitrate reductase, and proposes a novel method for preparing halophilic archaea nitrate reductase. Compared with previously reported methods for preparing halophilic archaea nitrate reductase, this method has simpler purification steps and yields nitrate reductase with higher purity.

[0030] (2)Nas Hor It possesses excellent enzymatic properties and exhibits superior adaptability to temperature, salinity, and pH, particularly for Fe... 3+ K + It exhibits varying degrees of tolerance to ethanol, glycerol, and isopropanol. A novel halophilic archaea nitrate reductase, Nas, possesses excellent traits. Hor It can be applied to the degradation of nitrates in pickled vegetables, sewage, etc. Attached Figure Description

[0031] Figure 1 This is a diagram for verifying the double enzyme digestion of the recombinant plasmid.

[0032] Figure 2 Nas after nickel column purification Hor The polyacrylamide gel electrophoresis analysis diagram.

[0033] Figure 3 For temperature versus Nas Hor Effect of catalytic activity (graph).

[0034] Figure 4 NaCl concentration versus Nas Hor The effect of catalytic activity is shown in the figure.

[0035] Figure 5 pH for Nas Hor The effect of catalytic activity is shown in the figure.

[0036] Figure 6 For Nas Hor Stability at different temperatures.

[0037] Figure 7 For Nas Hor Stability at different NaCl concentrations.

[0038] Figure 8 For Nas Hor Stability at different pH levels.

[0039] Figure 9 To test the effects of different organic solvents on Nas Hor The effect of catalytic activity is shown in the figure.

[0040] Figure 10 For different metal ions to Nas HorThe effect of catalytic activity is shown in the figure.

[0041] Figure 11 For Nas Hor Enzyme kinetics curves using nitrate as a substrate. Detailed Implementation

[0042] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0043] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0044] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0045] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0046] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0047] The halophilic archaea (Halorarius litoreus BND22) of this invention T Purchased from China General Microbiological Culture Collection Center, culture number CGMCC 1.18780.

[0048] The prokaryotic host was *Haloferax volcanii*, purchased from the Japan Culture Collection (JCM 8879). Based on strain *Haloferax volcanii*, a mutant strain was constructed, ultimately obtaining a uracil-thymidine auxotrophic mutant, denoted as *Haloferax volcanii* H1424. The construction method is well-known and can be found in: Allers T, Ngo HP, Mevarech M, et al. Development of Additional Selectable Markers for the Halophilic Archaeon Haloferax volcanii Based on the leuB and trpA Genes[J]. Applied and Environmental Microbiology, 2004, 70(2):943-953.

[0049] Example 1:

[0050] halophilic archaea nitrate reductase nas Hor Gene cloning;

[0051] (1) Obtaining the target gene

[0052] The inoculation loop was dipped into 30 μL of ddH2O and boiled at 100 °C for 10 min to obtain the DNA template. The target gene was amplified, and the PCR product detected by agarose gel electrophoresis was excised and recovered. The DNA gel recovery kit was then used for purification.

[0053] Primer BND22_2751-F-EcoRI: 5'-CGCGAATTCTCCGAGCGCACGAACGAG-3';

[0054] BND22_2751-R-BamHI: 5'-ATAGGATCCGTTGTCGGCGGCGGCGAC-3'.

[0055] PCR amplification system (25 μL):

[0056]

[0057] PCR amplification procedure:

[0058]

[0059] (2) Enzyme digestion of the target gene and vector

[0060] Using QuickCutTM The target gene fragment and the pTA04 vector were double-digested using EcoRI and BamHI (Takara) restriction enzymes at 37°C for 30 min. The digestion products were then purified again using a DNA gel extraction kit.

[0061] Enzyme digestion system (100 μL):

[0062]

[0063] (3) Construction of recombinant plasmids

[0064] The purified target gene from step (2) was ligated with the pTA04 vector at 16°C for 1-2 hours.

[0065] Connection system (10 μL):

[0066]

[0067]

[0068] (4) Transformation of recombinant plasmid into Escherichia coli DH5α

[0069] Add the 10 μL ligation system described above to a 1.5 mL Eppendorf tube containing 100 μL of E. coli DH5α competent cells. After mixing thoroughly, place the tube in an ice-water mixture for 30 min, then heat-shock it in a 42°C water bath for 90 s. Remove the tube and place it in an ice-water mixture for 2 min. Finally, add 500 μL of LB liquid medium and incubate with shaking for 45 min (37°C, 180 rpm). After incubation, centrifuge at 6000 rpm for 3 min, discard 500 μL of supernatant, and spread the remaining bacterial culture onto LB (ampicillin-containing) agar plates. Incubate overnight (12–15 h) at 37°C.

[0070] (5) Plasmid extraction and validation

[0071] Positive transformants that were verified by PCR were cultured in 3 mL of LB (containing ampicillin) liquid medium. Plasmids were extracted using a plasmid DNA mini-extraction kit and digested with enzymes for verification. The verification system was the same as shown in (1), and the enzyme digestion conditions were the same as in (2).

[0072] Enzyme digestion system (10 μL):

[0073]

[0074] The enzyme digestion products were detected by agarose gel electrophoresis, such as... Figure 1The diagram shown is a verification image of double enzyme digestion of the recombinant plasmid. Lanes 1 and 2 in the diagram represent the results of double enzyme digestion of the plasmid, and lane M represents the DNA marker. The successfully constructed plasmid was stored at -20℃ for subsequent experiments.

[0075] Example 2:

[0076] halophilic archaea nitrate reductase Nas Hor Heterogeneous expression;

[0077] The strain Haloferax volcanii H1424 was inoculated into Hv-YPC liquid medium (with thymine added at a concentration of 40 μg / mL). After growing to the exponential phase, it was converted into protoplasts. The recombinant plasmid was transformed into Haloferax vocanii H1424 by PEG-mediated transformation and cultured for about one week. The light red transformants were picked and verified by PCR.

[0078] Positive transformants that were verified by PCR were inoculated into 3 mL of Hv-YPC liquid medium and cultured at 37°C and 180 rpm for 3 days. Then, they were inoculated into 200 mL of Hv-YPC liquid medium at a 1% (v / v) inoculation rate to expand the culture. After the culture reached the stationary phase under the above conditions, the bacterial cells were collected by centrifugation and stored at -20°C.

[0079] Example 3:

[0080] halophilic archaea nitrate reductase Nas Hor Purification;

[0081] (1) After resuspending the bacterial cells in 20 mL of cell lysis buffer, sonicate until the bacterial cells are clear, and collect the supernatant by centrifugation for subsequent purification.

[0082] (2) After draining the preservation liquid (20% ethanol) from the nickel column, wash the column with 10 times the column bed volume of ddH2O and equilibrate the column with 10 times the column bed volume of cell lysis buffer.

[0083] (3) Pass the supernatant through the chromatography column twice (each time for 10 min in the column).

[0084] (4) Wash away the impurities with 25 mL of buffer I.

[0085] (5) Wash the column with 10 mL of buffer II, collect one tube of elution buffer per 1 mL, and store at 4 °C.

[0086] The eluent was analyzed by SDS-PAGE and enzyme activity was determined.

[0087] Figure 2The middle lanes are as follows: Lane M: protein pre-staining marker; Lane 1: supernatant after centrifugation; Lane 2: effluent after passing through the nickel column; Lane 3: tube 5 eluted with 40 mM imidazole; Lanes 4-9: tubes 1, 3, 5, 6, 7, and 9 eluted with buffer II.

[0088] (6) The column was washed sequentially with 10 column bed volumes of buffer (1M imidazole, 2M NaCl, 20mM Tris-HCl, pH 8.0) and 10 column bed volumes of ddH2O, respectively; finally, the column was immersed in 20% ethanol and stored at 4°C.

[0089] Example 4:

[0090] Ultrafiltration concentration;

[0091] The eluent obtained in Example 3 was concentrated to 1 mL using an ultrafiltration tube with a molecular cutoff of 10 kDa to obtain high-purity halophilic archaea nitrate reductase, denoted as Nas. Hor .

[0092] Example 5

[0093] Enzyme activity assay;

[0094] (1) Determining protein concentration

[0095] Protein concentration was determined using the Bradford Protein Assay Kit according to the instructions.

[0096] (2) Determination of enzyme activity

[0097] The 250 μL reaction system included 1 M NaCl, 4 mM methyl viologen (MV), 35 mM NaNO3, 17 mM Na2S2O4 (dissolved in 0.1 M NaHCO3), 7 μg enzyme, and 120 mM Na2CO3 / NaHCO3 solution (pH 9.0). Three experimental groups and three control groups were set up. The experimental groups received enzyme solution (Na2S2O4 / NaHCO3). Hor The control group was treated with an equal volume of buffer solution, and the reaction was terminated by shaking for 1 minute after reacting in a 60°C water bath for 20 minutes. 200 μL of 10 g / L sulfonamide was added to the reaction system, and the mixture was stirred for 4 minutes. Then, 200 μL of 1 g / L naphthylethylenediamine hydrochloride was added, and the mixture was stirred and reacted for 30 minutes in the dark. The OD value was measured at 540 nm. The activity of nitrate reductase can be calculated using the following formula.

[0098]

[0099] Y: Enzyme activity (U)

[0100] X: Nitrite content (mM) after enzyme reaction

[0101] Enzyme activity (U) is defined as the amount of enzyme required to produce 1 μg of nitrite per minute (μg / min).

[0102] Enzyme activity (U / mg) is defined as the number of enzyme activity units (μg / min / mg) per unit weight (mg) of protein.

[0103] The results showed that the specific enzyme activity measured under the conditions of 60℃, 1M NaCl and pH 9.0 was 25.14 μg / min / mg.

[0104] Example 6

[0105] Analysis of optimal reaction conditions;

[0106] (1) The optimal reaction temperature was determined in the range of 50–90℃;

[0107] The reaction temperatures were set at 50, 55, 60, 65, 70, 75, 80, 85, and 90 °C, with other conditions the same as in Example 5, and the enzyme activity of nitrate reductase was measured. The results showed that the optimal reaction temperature for this enzyme was 70 °C. Figure 3 ).

[0108] (2) The optimal NaCl concentration for the reaction was determined in the range of 0–4 M;

[0109] The final NaCl concentrations in the reaction system were 0, 0.25, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, and 4.0 M, and the reaction was carried out at the optimal temperature and pH 9.0. The activity of nitrate reductase was measured at different NaCl concentrations. The results showed that the optimal NaCl concentration for this enzyme reaction was 2.0 M. Figure 4 ).

[0110] (3) The optimal reaction pH was determined in the range of 6.0 to 10.5;

[0111] The reaction system was supplemented with 0.1M Na₂HPO₄ / NaH₂PO₄ buffer at pH 6.0, 6.5, 7.0, and 7.5; 50mM Tris-HCl buffer at pH 7.5, 8.0, 8.5, and 9.0; and 120mM Na₂CO₃ / NaHCO₃ buffer at pH 9.0, 9.5, 10.0, and 10.5, respectively. The reaction was carried out at the optimal reaction temperature and optimal NaCl concentration. The activity of nitrate reductase at different pH values ​​was measured. The results showed that the optimal reaction pH for this enzyme was 9.0. Figure 5 ).

[0112] Nas HorThe optimal reaction conditions are 70℃, 2.0M NaCl and pH 9.0.

[0113] Example 7:

[0114] Stability analysis;

[0115] (1)Nas Hor The specific procedure for the thermal stability analysis was as follows: the enzyme solution was incubated in a water bath at 30, 50, 70, and 90°C for 0, 30, 60, and 90 min, respectively. Enzyme activity was measured at the optimal reaction temperature, optimal NaCl concentration, and optimal pH. The results showed that the enzyme activity remained essentially unchanged after incubation at 30, 50, 70, and 90°C for 90 min; furthermore, a slight increase in relative enzyme activity was observed after incubation at 70 and 90°C for different durations, indicating that the enzyme possesses good thermal stability. Figure 6 ).

[0116] (2)Nas Hor The specific procedure for NaCl stability analysis was as follows: the enzyme solution was added to buffer solutions containing 0, 2, and 4 M NaCl (with the pH at its optimum), and incubated at room temperature for 0, 30, 60, and 90 min. Enzyme activity was measured at the optimum reaction temperature. The results showed that the enzyme maintained good activity after incubation at 2 M NaCl for 0, 30, 60, and 90 min; however, the relative enzyme activity decreased over time when incubated at 0 and 4.0 M NaCl concentrations. In summary, the relative enzyme activity remained at 70% or higher after incubation at 0, 2.0, and 4.0 M NaCl for 0–90 min, demonstrating good NaCl stability. Figure 7 ).

[0117] (3)Nas Hor The specific procedure for pH stability analysis was as follows: The enzyme solution was added to buffers at pH 6.0 (Na₂HPO₄ / NaH₂PO₄ buffer), pH 8.0 (Tris-HCl buffer), and pH 10.0 (Na₂CO₃ / NaHCO₃ buffer), and incubated at room temperature for 0, 30, 60, and 90 min. Enzyme activity was measured at the optimal reaction temperature and optimal NaCl concentration. The results showed that the enzyme maintained better activity when incubated in buffers at pH 8.0 and 10.0; when incubated in buffer at pH 6.0, the relative enzyme activity showed a decreasing trend with increasing incubation time, reaching a minimum of approximately 60%. Compared to incubation at pH 6.0, the enzyme exhibited better stability when incubated at pH 8.0 and 10.0. Figure 8 ).

[0118] Example 8:

[0119] Resistance analysis to organic solvents and metal ions;

[0120] (1) The specific operation of the organic solvent tolerance analysis is as follows: 15% (v / v) of organic solvents are added to the reaction system: methanol, ethanol, acetonitrile, acetone, glycerol, isopropanol, DMSO (dimethyl sulfoxide), DMF (N,N-dimethylformamide); surfactants: Tween 20, Tween 80, Triton X-100.

[0121] Enzyme activity was measured at the optimal reaction temperature, NaCl concentration, and pH. The results showed that ethanol, glycerol, and isopropanol had varying degrees of promoting effects on the enzyme, with ethanol and glycerol exhibiting the most significant promoting effects. Methanol, acetonitrile, acetone, DMSO, DMF, Tween 20, Tween 80, and Triton X-100 showed varying degrees of inhibitory effects on the enzyme. DMF, Tween 20, Tween 80, and Triton X-100 achieved inhibition rates exceeding 80%, while acetone showed the lowest inhibition rate, approximately 20%. Figure 9 ).

[0122] (2) The specific procedure for metal ion tolerance analysis is as follows: Add Ca to the reaction system at a final concentration of 5 mM. 2+ 、Sr 2 + Zn 2+ Fe 3+ K + Cu 2+ Ni + Mg 2+ Ba 2+ Enzyme activity was determined at the optimal reaction temperature, NaCl concentration, and pH.

[0123] Figure 10 For different metal ions to Nas Hor Effect of catalytic activity diagram;

[0124] The results showed that Fe 3+ and K + Ni has a promoting effect on this enzyme; the other seven metal ions have varying degrees of inhibitory effects on this enzyme; among them, Ni... 2+ and Ba 2+ The inhibitory effect is the strongest, with an inhibition rate of approximately 60%. (Ca) 2+ 、Sr 2+ and Zn 2+ The inhibition rate was approximately 40%. This indicates that the enzyme has a certain degree of tolerance to metal ions.

[0125] Example 9:

[0126] Enzyme kinetic parameters;

[0127] In the reaction system, NaNO3 was used as the substrate at concentrations of 0, 5, 10, 15, 20, 25, 30, 35, and 40 mM. Enzyme activity was measured at the optimal reaction temperature, NaCl concentration, and pH.

[0128] Figure 11 For Nas Hor Enzyme kinetics curves using nitrate as a substrate;

[0129] Enzyme kinetic curves were constructed using Origin 2015 software, from which the V of the enzyme can be determined. max The value was 30.94 μg / min / mg, K m The value is 1.64mM ( Figure 11 ).

[0130] Note: The above embodiments are only used to illustrate the present invention and are not intended to limit the technical solutions described in the present invention. Therefore, although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the present invention. All technical solutions and improvements that do not depart from the spirit and scope of the present invention should be covered within the scope of the claims of the present invention.

[0131] Nas Hor Amino acid sequence: SEQ ID No. 1

[0132] MSERTNEPRESVCPFCGVGCGLTYDAERGKGVGWRGAVNTRGELCPKGVAAFEHVDHADRLRTPLIREDGALREATWDEALDRIETAFGEAIARHGPDAAFFFASSKCTNEANYVVQKLARALGTNNVDNCARLCHASTVAAMAERFGAGAMTNSLRDLTEADVFLVAGANPAVNHPVIFRSYLLPALKAGTELIHLDPRATATTDAATHHLPVRPGFDIPLLNAMAAVVVEEGLVDEAFVRERTTGFEALRAFLDEVDVDAAAEQTGVDAETLRTAARSYGSADRAAIFTGMGLSQHHCGTDNVHALLNLALLTGNVGTRGAGVNPLRGQNNVQGAGDVGALPDRLPGGRIDDPATHERCATEWGFDVPTTPGLTEVEATHRFGEDVRVAYVFGENPAVTEPNVSRVREKLASLDCLVVHDVVRSETVEFADVVLPGGTWAETDGTVTNTDRQVQRMRAMADPPGDARRDLDVLGAVAERLVGEGFACDGPEAVFEELTRVSATYAGMSYDGIGTGSQRWPFPEGATEGTAVLHEGTFASGSTTADLRIVEHVDPVDPVGDDELVLTTGRILEHYNSGALTRRSGTLVRLRGTDALQIHPGDAARRGIEDGDRVRVANDRGMVELAAEVTPGVRSGTVFCTFHFADPLVNTLTGDTLDPVAKIPPFKHSAVRVTVAAADN

[0133] Nas Hor Nucleotide sequence: SEQ ID No.2

[0134]

Claims

1. The application of a halophilic archaea nitrate reductase in the degradation of nitrates in pickled vegetables and wastewater, characterized in that, The steps of the halophilic archaea nitrate reductase are as follows: (1) Construction of recombinant plasmid pTA04- nas Hor ; Based on halophilic archaea ( Halorarius litoreus BND22 T Functional alignment of the whole genome sequence of a halophilic archaea yielded a nitrate reductase gene. nas Hor Then, the target gene fragment was amplified using PCR technology. nas Hor Then, through molecular cloning technology, nas Hor The recombinant plasmid pTA04- was constructed by ligating it into the pTA04 vector. nas Hor The halophilic archaea were purchased from the China General Microbiological Culture Collection Center, with the culture accession number CGMCC 1.18780. (2) Recombinant plasmid pTA04- nas Hor Transformation of prokaryotic hosts and expression; The recombinant plasmid pTA04- constructed in step (1) nas Hor Recombinant cells were obtained by transforming a prokaryotic host and inoculated into Hv-YPC liquid medium. After culturing to the stable growth phase, the cells were collected by centrifugation and stored for later use. The prokaryotic host was based on the bacterial strain. Haloferax volcanii The mutant strain was constructed, and the uracil-thymidine auxotrophic mutant strain was finally obtained, denoted as... Haloferax volcanii H1424; Haloferax volcanii Purchased from the Japan Microbial Culture Collection Center, culture accession number JCM 8879; (3) Purification of nitrate reductase from halophilic archaea; The bacterial cells collected in step (2) were resuspended in cell lysis buffer. After resuspending, the cells were sonicated to disrupt the cell structure. The supernatant was collected after centrifugation and purified using nickel affinity chromatography. Subsequently, the enzyme solution was concentrated by ultrafiltration to obtain high-purity halophilic archaea nitrate reductase, denoted as Nas. Hor Its nucleotide sequence is shown in SEQ ID NO.2, and its amino acid sequence is shown in SEQ ID NO.

1.

2. The use of the halophilic archaea nitrate reductase according to claim 1 in the degradation of nitrates in pickled vegetables and wastewater, characterized in that, The Hv-YPC culture medium in step (2) consists of the following components per liter: yeast extract 5.0 g, soybean peptone 1.0 g, acid-hydrolyzed casein 1.0 g, KCl 4.2 g, MgSO4·7H2O 33.0 g, MgCl2·6H2O 30.0 g, NaCl 144.0 g, 1 M, pH 8.0 Tris-HCl 12.0 mL, and CaCl2 0.33 g. The above components are diluted to 1 L with distilled water, the pH is adjusted to 7.5, and the medium is sterilized at 115 °C for 30 min.

3. The use of the halophilic archaea nitrate reductase according to claim 1 in the degradation of nitrates in pickled vegetables and wastewater, characterized in that, The culture conditions described in step (2) are 37 ℃, 200 rpm, and culture for 3 to 4 days.

4. The use of the halophilic archaea nitrate reductase according to claim 1 in the degradation of nitrates in pickled vegetables and wastewater, characterized in that, The preservation method described in step (2) is low-temperature preservation at a temperature of -20 ℃.

5. The use of the halophilic archaea nitrate reductase according to claim 1 in the degradation of nitrates in pickled vegetables and wastewater, characterized in that, The cell lysis buffer in step (3) consists of 2 M NaCl, 20 mM Tris-HCl, and pH 8.

0.

6. The use of the halophilic archaea nitrate reductase according to claim 1 in the degradation of nitrates in pickled vegetables and wastewater, characterized in that, The steps of nickel column affinity chromatography purification in step (3) are as follows: drain the 20% ethanol stored in the nickel column, wash the column with 10 column volumes of ddH2O, and then equilibrate the column with 10 column volumes of cell lysis buffer; flow the supernatant collected by centrifugation into the nickel column and let it stand for 10 min; elute the impurities with 10 column volumes of buffer I; and elute the target protein with 5 column volumes of buffer II. The components of buffer I are 40 mM imidazole, 2 M NaCl, 20 mM Tris-HCl, pH 8.0; The buffer II consisted of 100 mM imidazole, 2 M NaCl, 20 mM Tris-HCl, and pH 8.

0.

7. The use of the halophilic archaea nitrate reductase according to claim 1 in the degradation of nitrates in pickled vegetables and wastewater, characterized in that, In step (3), ultrafiltration is performed using an ultrafiltration centrifuge tube with a molecular weight cutoff of 10 kDa for concentration.

Images